Functional Characterization of Negri Bodies (NBs) in Rabies

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癫痫患者脑电图异常和血清BDNF_GFAP_miR-7-5p_表达特征及与认知功能的关系

入关节内进行操作㊂而小切口手术则是在关节外进行,通过在皮肤上开一个较大的切口直接暴露关节外的结构进行操作㊂因此,小切口手术可能对肩关节内的结构干扰更少,从而减少了对肩关节功能的影响㊂此外,关节镜下手术需要建立关节腔与外界的通道,这个过程中会对关节周围的组织和韧带造成一定的损伤㊂而小切口手术虽然需要在皮肤上开一个较大的切口,但由于是在关节外进行操作,对关节内部结构的损伤更小,因此可能对肩关节功能恢复更有利㊂另外,与关节镜下高位关节内LHBT切断固定治疗相比较,小切口低位关节外结节间沟下LHBT切断固定治疗伴有肱二头肌长头肌腱炎SIS患者,可更有效提高临床治疗疗效[8]㊂研究结果显示,观察A组临床治疗总有效率80.00%,与观察B组95.74%比较,明显较低㊂原因可能是对于伴有肱二头肌长头肌腱炎SIS患者,关节镜下高位关节内LHBT切断固定治疗是比较常规的治疗方式,但在手术操作过程中可能会导致一些问题㊂比如,关节镜下高位关节内LHBT切断固定治疗操作难度较大,因为需要在关节镜下完成,所以对医生的操作水平要求较高㊂此外,由于是在关节内进行手术,术后可能会产生一些并发症,如关节肿胀㊁疼痛等㊂相比之下,小切口低位关节外结节间沟下LHBT切断固定治疗伴有肱二头肌长头肌腱炎SIS患者则具有一些优势㊂首先,手术操作相对简单,切口较小,术后恢复较快,减少了患者的痛苦㊂其次,手术时间较短,降低了患者的经济负担㊂最后,小切口低位关节外结节间沟下LHBT切断固定治疗术后并发症较少,降低了患者的风险㊂综上所述,与关节镜下高位关节内LHBT切断固定治疗相比较,小切口低位关节外结节间沟下LHBT 切断固定治疗伴有肱二头肌长头肌腱炎SIS患者,可更有效促进肩关节功能恢复,提高临床治疗疗效㊂ʌ参考文献ɔ[1]㊀Kooistra B,Gurnani N,Weening A,et al.Tenotomy or teno-desis for tendinopathy of the long Head of the biceps brachii:an updated systematic review and meta-analysis.[J].Ar-throsc Sports Med Rehabil,2021,3(4):1199-1209. [2]㊀Hughes JD,Gibbs CM,Drummond M,et al.Failure rates andclinical outcomes after treatment for long-head bicepsbrachii tendon pathology:a comparison of three treatmenttypes.[J].JSES Int,2021,5(4):630-635.[3]㊀曲博,石向明,王成健,等.MSCT联合MRI在肩关节损伤诊断及损伤程度判定方面的价值[J].河北医科大学学报,2024,45(1):35-39.[4]㊀龚礼,周明,范少勇,等.关节镜下两种肌腱固定治疗肱二头肌长头近端腱损伤的比较[J].中国组织工程研究, 2023,27(28):4533-4538.[5]㊀任晔,祁军,游洪波,等.两种手术方式治疗高龄肩袖损伤合并肱二头肌腱长头腱损伤疗效比较[J].实用骨科杂志,2021,27(7):577-582.[6]㊀吴晓翔,郑卫丛,常乾震,等."筋骨平衡,以筋为先"理论在肩峰下撞击综合征中的应用探讨[J].山东中医杂志, 2023,42(5):442-447.[7]㊀雷博艺,周百刚,冯宏伟,等.关节镜下单纯切断术与切断固定术治疗肱二头肌长头肌腱炎的临床疗效及安全性比较[J].检验医学与临床,2022,19(23):3279-3282. [8]㊀高秋明,丁浩亮,孙健.肩关节镜下肱二头肌长头肌腱转位重建肩关节上关节囊修复巨大肩袖撕裂[J].中华创伤杂志,2021,37(3):222-228.ʌ文章编号ɔ1006-6233(2024)04-0623-07癫痫患者脑电图异常和血清BDNF GFAP miR-7-5p表达特征及与认知功能的关系白㊀杨,㊀杨㊀越,㊀齐会珍(新疆医科大学第五附属医院神经内科,㊀新疆㊀乌鲁木齐㊀830000)ʌ摘㊀要ɔ目的:探究癫痫患者脑电图异常和BDNF㊁GFAP㊁miR-7-5p表达特征,并探究各指标与患者认知功能的关系㊂方法:本研究以我院2017年4月至2021年4月期间治疗的294例癫痫患者为对象,采用MoCA评分对患者的认知功能进行评价,并据此对患者进行分组:将得分ȡ26分的198例患者纳入认知正常组,得分<26分的96例患者纳入认知障碍组,患者均接受脑电图检查㊁血清BDNF㊁GFAP㊁miR-7-5p检测,比较组间脑电图检查异常情况及血清BDNF㊁GFAP㊁miR-7-5p表达差异,并探究上述指标与认知障碍的关联㊂结果:对两组患者的MoCA评分进行分析可知认知障碍组患者的Mo-㊃326㊃ʌ基金项目ɔ新疆维吾尔自治区自然科学基金资助项目,(编号:2023D01C246)CA各项评分均低于认知正常组(P<0.05)㊂与认知正常组患者相比,认知障碍组患者脑电图无异常占比降低,认知障碍组患者脑电图痫样放电㊁脑电图慢波发放的发生率高于认知正常组(P<0.05)㊂与认知正常组患者相比较,认知障碍组BDNF㊁miR-7-5p降低,GFAP升高(P<0.05)㊂通过Pearson相关性分析发现,患者的MoCA评分与血清中的BDNF和miR-7-5p表达呈正相关,与其GFAP表达呈负相关(P<0.05)㊂经ROC曲线分析,血清BDNF㊁miR-7-5p及GFAP表达对癫痫患者认知障碍的发生具有一定诊断价值,其AUC值分别为0.836㊁0.845㊁0.957㊂结论:伴有认知障碍的癫痫患者脑电图异常特征以脑电图痫样放电㊁脑电图慢波发放为主,血清BDNF㊁miR-7-5p表达低于认知正常的癫痫患者,GFAP 表达高于认知正常的癫痫患者,且血清BDNF㊁miR-7-5p及GFAP表达对癫痫患者认知障碍的发生具有一定诊断价值㊂ʌ关键词ɔ㊀癫㊀痫;㊀脑电图异常;㊀认知障碍;㊀脑源性神经营养因子;㊀胶质纤维酸性蛋白;㊀miR-7-5pʌ文献标识码ɔ㊀A㊀㊀㊀㊀㊀ʌdoiɔ10.3969/j.issn.1006-6233.2024.04.018The Relationship Between EEG Abnormalities and Serum BDNF GFAP miR-7-5p Expression Characteristics and Cognitive Function in Epilepsy PatientsBAI Yang,YANG Yue,QI Huizhen(The Fifth Affiliated Hospital of Xinjiang Medical University,Xinjiang Urumqi830000,China)ʌAbstractɔObjective:To explore the relationship between EEG abnormalities,serum levels of Brain-Derived Neurotrophic Factor(BDNF),Glial Fibrillary Acidic Protein(GFAP),and miR-7-5p expression characteristics,and cognitive function in epilepsy patients.Methods:The study included294epilepsy pa-tients treated at our hospital from April2017to April2021.Cognitive function was assessed using the Montreal Cognitive Assessment(MoCA)scale.Patients were divided into two groups based on their MoCA scores:198 patients with scoresȡ26were included in the cognitively normal group,while96patients with scores<26 were included in the cognitive impairment group.All patients underwent EEG examinations.The study com-pared EEG abnormalities and serum levels of BDNF,GFAP,and miR-7-5p between the groups and investi-gated their association with cognitive impairment.Results:Analysis of the MoCA scores showed that patients in the cognitive impairment group had significantly lower scores than those in the cognitively normal group(P <0.05).Compared to the cognitively normal group,patients in the cognitive impairment group had a lower proportion of normal EEGs and higher rates of epileptiform discharges and slow-wave activity(P<0.05).Ser-um levels of BDNF and miR-7-5p were lower,while GFAP levels were higher in the cognitive impairment group(P<0.05).Pearson correlation analysis indicated that cognitive function in epilepsy patients was posi-tively correlated with BDNF and miR-7-5p levels and negatively correlated with GFAP levels(P<0.05). ROC curve analysis demonstrated that BDNF,miR-7-5p,and GFAP levels have diagnostic value for cogni-tive impairment in epilepsy patients,with AUC values of0.836,0.845,and0.957,respectively.Conclu-sion:The abnormal characteristics of EEG in patients with cognitive impairment are mainly EEG discharge and slow wave distribution of EEG.The expression of serum BDNF and miR-7-5p is lower than that of patients with normal epilepsy,and the expression of GFAP is higher than that of patients with normal epilepsy,and se-rum BDNF,miR-7-5p and GFAP have certain diagnostic value for the occurrence of cognitive impairment in patients with epilepsy.ʌKey wordsɔ㊀Epilepsy;㊀EEG abnormalities;㊀Cognitive impairment;㊀Brain-derived neurotrophic factor;㊀Glial fibrillary acidic protein;㊀miR-7-5p㊀㊀癫痫是一种以反复无预兆的脑神经元异常放电为特征的慢性脑部疾病,对全球数百万人造成显著影响[1]㊂除了发作性症状外,许多癫痫患者还表现出不同程度的认知功能障碍,这对患者的生活质量造成了㊃426㊃严重影响[2]㊂目前,癫痫的诊断主要依赖于脑电图(Electroencephalogram,EEG)捕捉到的异常放电㊂然而,脑电图并不能全面揭示癫痫患者认知功能障碍的生物学基础[3]㊂近年来,血清生物标志物,如脑源性神经营养因子(Brain-Derived Neurotrophic Factor,BD-NF)㊁胶质纤维酸性蛋白(Glial Fibrillary Acidic Pro-tein,microRNA,GFAP)和microRNA(如microRNA-7-5p,miR-7-5p)在神经系统疾病中的作用日益受到关注㊂BDNF在神经系统中是关键的神经营养因子,影响神经元生存㊁发育和认知功能,其功能异常与多种神经退行性和精神疾病有关㊂而GFAP作为星形胶质细胞的标志,参与脑组织结构维持和神经炎症反应,其表达水平的变化与多种神经疾病相关㊂miR-7-5p则是调控基因表达的microRNA之一,对神经发育㊁突触功能和神经保护有重要影响,其异常表达与多种神经系统疾病相关[4-5]㊂本研究旨在探究这些生物标志物在癫痫患者中的表达模式,以及它们与患者脑电图异常和认知功能障碍之间的关系,从而为癫痫的诊断和治疗提供新的见解,现将结果报道如下㊂1㊀资料与方法1.1㊀一般资料:本研究以我院2017年4月至2021年4月期间治疗的294例癫痫患者为对象,采用MoCA 评分对患者的认知功能进行评价,并据此对患者进行分组:将得分ȡ26分的198例患者纳入认知正常组,得分<26分的96例患者纳入认知障碍组,两组一般资料比较差异无统计学意义(P>0.05),见表1㊂纳入标准:①年龄在18~65岁;②根据国际癫痫学会的标准(临床出现两次非诱发性癫痫发作,间隔至少24h),确诊为癫痫;③能够完成标准的认知功能评估测试;④无严重的精神疾病,可进行认知评估;⑤患者或其法定代理人知情同意㊂排除标准:①患神经退行性疾病,如阿尔茨海默病㊁帕金森病;②严重的精神疾病,如精神分裂症㊁严重的抑郁症或焦虑症;③有药物滥用史或酒精依赖者;④近期进行过脑部手术或有脑部外伤性损伤;⑤严重全身性疾病,如未控制的糖尿病㊁高血压㊁心脏病㊂表1㊀两组一般资料比较[ xʃs,n(%)]组别认知正常组(n=198)认知障碍组(n=96)χ2/t P 性别男123(62.12)65(67.71)0.8750.349女75(37.88)31(32.29)BMI(kg/m2)22.45ʃ2.0222.16ʃ1.98 1.1620.246发作持续时间(min) 1.26ʃ0.64 1.34ʃ0.71-0.9690.333高血压18(9.09)9(9.38)0.0060.937病程(年) 4.56ʃ1.45 4.79ʃ1.64-1.2210.223癫痫发作频率(次/3月) 6.78ʃ1.56 6.89ʃ1.78-0.5410.589癫痫家族史31(15.66)18(18.75)0.4450.505糖尿病28(14.14)15(15.63)0.1140.7361.2㊀方法:脑电图检查:使用美国Nicolet公司生产的32通道全数字化视频脑电图监测系统,电极的放置遵循国际公认的10/20系统标准,连续进行24h的脑电图描记㊂血清BDNF㊁GFAP表达:在空腹状态下,从每个组别的成员抽取5mL静脉血,然后以3000r/min的速度离心10min,以此来获取血清样本㊂然后通过酶联免疫吸附试验(ELISA)方法,对BDNF和GFAP的水平进行检测,所用试剂盒由上海信裕生物工程有限公司提供㊂miR-7-5p表达:使用北京凯诗源生物科技有限公司的Trizol试剂盒从血清中提取总RNA,并检查其完整性㊂接着,采用上海恒斐生物科技有限公司的反转录试剂盒,将提取的RNA转录成cDNA㊂最后,通过美国ABI公司的7900型qRT-PCR仪,使用实时荧光定量PCR方法,对血清中的miR-7-5p的表达水平进行测定㊂在qRT-PCR反应体系中,每个反应包括0.5μL的上下游引物,2μL的cDNA,10μL的SYBR Green I qPCR Master Mix(由上海远慕生物科技有限公司提供),0.4μL的50倍浓缩ROX Reference㊃526㊃Dye,以及6.6μL的双蒸水㊂miR-7-5p的上游引物为ATGACGAGGTGAGTGCACG;下游引物为GCAGATC-CACATGCTCTGGA㊂PCR反应结束后,收集数据并分析CT值(循环阈值)㊂miR-7-5p的表达水平,以U6作为内参,通过2-ΔΔCT法计算miR-7-5p的相对表达量㊂1.3㊀观察指标:采用MoCA量表[6]对患者的认知功能进行评价,并比较组间差异㊂患者均接受脑电图检查,比较组间脑电图检查异常情况㊂检测比较组间患者血清BDNF㊁GFAP及miR-7-5p表达,并对上述指标与认知障碍的相关性及其诊断价值进行分析㊂1.4㊀统计学方法:使用SPSS23.0软件进行数据处理,患者性别㊁既往病史㊁家族癫痫史等计数资料以n(%)表示,均进行卡方检验;等级资料如患者的脑电图异常情况进行秩和检验;计量资料如年龄㊁病程㊁癫痫发作频率及时间等以( xʃs)表示,组间进行独立样本t检验;采用Pearson相关性分析探究患者的认知能力与各指标间的相关性;采用ROC曲线探究上述指标对患者认知障碍的诊断价值㊂α=0.05㊂2㊀结㊀果2.1㊀MoCA评分比较:在不同的认知维度方面,认知正常组的平均得分均明显高于认知障碍组(P<0.05)㊂见表2㊂表2㊀MoCA评分比较( xʃs,分)组别n命名能力视空间和执行能力抽象能力延迟回忆注意力语言能力定向力总分认知正常组198 2.99ʃ0.56 4.55ʃ0.46 1.44ʃ0.29 4.56ʃ0.79 5.54ʃ0.98 2.44ʃ0.49 5.87ʃ0.9927.39ʃ2.46认知障碍组96 2.67ʃ0.67 3.66ʃ0.78 1.33ʃ0.45 3.05ʃ0.58 4.11ʃ0.79 1.66ʃ0.61 5.44ʃ0.8521.92ʃ0.68 t 4.30312.260 2.52616.67012.46411.789 3.65221.377 P<0.001<0.0010.012<0.001<0.001<0.001<0.001<0.0012.2㊀脑电图异常情况比较:与认知正常组患者相比,认知障碍组患者脑电图无异常占比降低,认知障碍组患者脑电图痫样放电㊁脑电图慢波发放的检出率不同于认知正常组(P<0.05)㊂见表3㊂表3㊀脑电图异常情况比较n(%)组别n脑电图无异常脑电图痫样放电脑电图慢波发放认知正常组198128(64.65)19(9.60)51(25.76)认知障碍组968(8.33)55(57.29)33(34.38) Z104.436P<0.0012.3㊀血清BDNF㊁miR-7-5p㊁GFAP表达比较:与认知正常组患者相比较,认知障碍组BDNF㊁miR-7-5p降低,GFAP升高(P<0.05)㊂见表4㊂表4㊀血清BDNF miR-7-5p GFAP表达比较( xʃs)组别n BDNF(ng/mL)miR-7-5p GFAP(ng/L)认知正常组1987.35ʃ1.55 1.03ʃ0.23 2.37ʃ0.49认知障碍组96 5.45ʃ1.230.74ʃ0.18 3.89ʃ0.78㊃626㊃t 10.51010.845-20.372P<0.001<0.001<0.0012.4㊀血清BDNF ㊁miR -7-5p ㊁GFAP 表达与MoCA 评分的相关性:通过Pearson 相关性分析发现,患者的MoCA 评分与血清中的BDNF 和miR -7-5p 表达呈正相关,与其GFAP 表达呈负相关(P <0.05)㊂见表5,图1~3㊂2.5㊀血清BDNF ㊁miR -7-5p 及GFAP 表达对癫痫患者认知障碍的诊断价值:经ROC 曲线分析,血清BD-NF ㊁miR -7-5p 及GFAP 表达对癫痫患者认知障碍的发生具有一定诊断价值,其AUC 值分别为0.836㊁0.845㊁0.957(P<0.05)㊂见表6㊁图4㊂表5㊀血清BDNF miR -7-5p GFAP 表达与MoCA 评分的相关性相关系数BDNF miR -7-5p GFAP r 0.4100.443-0.609P<0.001<0.001<0.001表6㊀血清BDNF miR -7-5p 及GFAP 水平对癫痫患者认知障碍的诊断价值指标AUC 最佳截断值95%CI下限㊀㊀㊀㊀上限灵敏度特异度P BDNF 0.836 6.6750.7900.8810.6970.833<0.001miR -7-5p0.8450.8750.8000.8910.7880.792<0.001GFAP0.9573.1450.9330.9810.9490.854<0.001图1㊀MoCA 评分与血清BDNF的散点图图2㊀MoCA 评分与血清miR -7-5p的散点图图3㊀MoCA 评分与血清GFAP的散点图图4㊀BDNF 、miR -7-5p 及GFAP 水平对癫痫患者认知障碍的诊断价值曲线㊃726㊃3㊀讨㊀论癫痫的核心问题在于脑神经元的异常放电,这可以由多种因素引起,包括神经递质的失衡㊁轴突的异常发芽或遗传变异㊂这些异常放电导致了癫痫的典型临床表现,如意识丧失㊁抽搐和全身痉挛㊂更为复杂的是,癫痫不仅仅是一系列独立的发作,大约40%的患者还会经历认知功能障碍,如记忆减退㊁注意力不集中和执行功能受损,这些问题进一步影响了患者的日常生活和社会参与㊂从流行病学的角度来看,癫痫的发病率正在全球范围内逐年上升,这可能与人口老龄化㊁诊断技术的改进和对疾病认知的提高有关㊂癫痫发作的突发性和不可预测性使得患者面临着较高的意外伤害风险,长期而言,癫痫对患者的心理健康㊁记忆㊁行为和认知能力可能产生深远影响㊂因此,癫痫不仅是一种医学上的挑战,也是一个需要社会和心理层面关注的问题㊂虽然目前有多种抗癫痫药物和治疗手段,但对于某些难治性癫痫患者来说,有效控制症状仍然具有挑战性㊂癫痫患者认知功能障碍的评估㊁治疗和干预是当前研究的热点,目的是改善患者的整体生活质量㊂而对于继发性癫痫来说,有研究表明高压氧配合单唾液酸四己糖神经节苷脂钠治疗继发性癫痫能够有效改善癫痫症状,促进免疫功能恢复,进而缓解机体氧化应激反应㊂本研究首先对两组患者的认知功能进行了评估,结果显示,认知正常组与认知障碍组之间存在显著差异㊂具体来说,认知正常组的成员在各项认知测试中表现得更好,得分更高,这反映出他们在记忆力㊁注意力㊁语言能力㊁执行功能等方面的认知能力较强㊂这种差异强调了认知功能评估在区分不同认知状态的重要性,同时也可能揭示了与认知障碍相关的生物标志物或疾病进程的存在㊂本研究观察到认知障碍组患者脑电图异常和痫样放电㊁慢波发放的发生率高于认知正常组,说明脑电图异常与癫痫患者认知功能障碍相关,这与先前Pinto LF等[7]与Babiloni C等[8]的研究一致㊂李陶乐等[9]的研究指出,在颞叶癫痫患者群体中,MRI揭示的责任病灶与脑电图所呈现的频繁癫痫放电㊁慢波活动和异常背景波之间存在显著联系,这些因素与患者在心理㊁情绪和认知功能上的受损紧密相关㊂脑电图的异常活动会干扰早期的靶刺激识别能力,并对晚期信息处理的整合产生影响,尤其是在认知功能方面,这种影响更加显著㊂另外,在脑电图表现异常的患者中,P3波的潜伏期延长和波幅降低,这暗示着他们在认知后期的高级编码处理能力受到损害,以及由于脑力资源的不足导致信息记忆能力的下降㊂而姚晓娟等[10]和朱慧等[11]的研究也表明,脑电图异常作为独立因素,与患者出现精神情绪障碍之间有关联,特别是慢波放电的范围与认知功能损害之间呈现正相关关系㊂本研究还发现,血清中BDNF和miR-7-5p的水平在认知障碍组中显著低于认知正常组,而GFAP水平则相反㊂BDNF作为一种关键的神经营养素,对神经系统的健康㊁认知功能以及感觉系统的维护具有密切的相关性,它在脑功能学习过程的多个层面中具有重要作用㊂在BDNF水平下降的情况下,神经元损伤的程度会增加,进而可导致记忆力和认知功能的减退㊂同时,闻红斌[12]的研究发现,癫痫患者的血清BDNF水平降低,并且伴随着严重的认知功能损害㊂这说明血清中BDNF的水平与患者认知功能损伤的程度之间存在一定的相关性,这一发现与本研究的部分结果是一致的㊂Xu H[13]的研究发现,在缺氧缺糖/复氧的大鼠模型中,miR-7-5p表达降低,而姜黄素能够通过调节miR-7-5p来改善神经损伤和认知功能障碍㊂另一方面,Li等[14]的研究表明,降低LncRNA MEG3的表达可以通过靶向miR-7-5p来缓解异氟醚治疗引起的认知功能障碍,并具有神经保护作用㊂结合这些研究结果,可以推测,在癫痫发作后,患者体内的应激反应导致中枢神经系统中的神经炎症增加,从而增加Ln-cRNA MEG3及炎症因子的表达㊂LncRNA MEG3通过靶向作用于miR-7-5p,参与调控神经炎症和氧化应激,对癫痫患者认知功能障碍的发生和发展起着重要作用㊂GFAP是星形胶质细胞活化的核心标志物, GFAP水平的升高促使星形胶质细胞在结构和功能上发生变化,这可能触发神经功能障碍,进而导致认知功能的下降㊂本研究的Pearson相关性分析结果显示血清BD-NF㊁miR-7-5p水平与癫痫患者认知功能呈正相关, GFAP水平与认知功能呈负相关㊂这些发现提示,这些生物标志物可能在癫痫相关认知功能障碍的发生和发展中起着重要作用㊂此外,通过ROC曲线分析发现,BDNF㊁miR-7-5p和GFAP的水平对癫痫患者认知障碍的诊断具有一定的价值㊂这表明,这些血清生物标志物不仅能够反映认知功能障碍的存在,还可能成为未来癫痫患者认知障碍的诊断标志物㊂综上所述,癫痫患者的脑电图异常㊁血清BDNF㊁GFAP和miR-7-5p水平与认知障碍的发生密切相关,且具有一定的诊断价值㊂ʌ参考文献ɔ[1]㊀Beghi E.The Epidemiology of epilepsy[J].Neuroepidemiolo-㊃826㊃gy ,2020,54(2):185-191.[2]㊀Katyayan A ,Diaz -Medina G.Epilepsy :epileptic syndromesand treatment [J ].Neurol Clin ,2021,39(3):779-795.[3]㊀Benbadis SR ,Beniczky S ,Bertram E ,et al.The role of EEGin patients with suspected epilepsy [J ].Epileptic Disord ,2020,22(2):143-155.[4]㊀王满利,章晓富,樊玉香,等.癫痫患者血清长链非编码RNA 母系表达基因3㊁miR -7-5p 表达与认知功能的关系[J ].中国卫生检验杂志,2022,32(24):2998-3002.[5]㊀Zhao J ,Wang B.MiR -7-5p enhances cerebral ischemia -reperfusion injury by degrading sirt1mRNA [J ].Cardiovasc Pharmacol ,2020,76(2):227-236.[6]㊀Pinto TCC ,Machado L ,Bulgacov TM ,et al.Is the montrealcognitive assessment (MoCA )screening superior to the Mini-Mental State Examination (MMSE )in the detection of mild cognitive impairment (MCI )and Alzheimer's Disease (AD )in the elderly ?[J ]Int Psychogeriatr ,2019,31(4):491-504.[7]㊀Pinto LF ,Adda CC ,Silva LC ,et al.Contralateral interictaland ictal EEG epileptiform activity accentuate memory im-pairment in unilateral mesial temporal sclerosis patients [J ].Neuropsychology ,2017,31(3):268-276.[8]㊀Babiloni C ,Del Percio C ,Lizio R ,et al.Cortical sources ofresting state electroencephalographic alpha rhythms deterio-rate across time in subjects with amnesic mild cognitive im-pairment [J ].Neurobiol Aging ,2014,35(1):130-142.[9]㊀李陶乐,许虹.运用事件相关电位评价颞叶癫痫患者认知功能及其与头颅磁共振和脑电图异常的相关性分析[J ].中华医学杂志,2019,99(27):2115-2118.[10]㊀姚晓娟,陈旨娟,毓青,等.脑电图-功能磁共振成像技术对局灶性癫痫致痫灶定位的价值[J ].中华医学杂志,2015,95(13):987-990.[11]㊀朱慧,肖正军,高茂军.癫痫患者事件相关电位P300及影响因素研究[J ].中国实用神经疾病杂志,2016(3):14-15.[12]㊀闻红斌.血清BDNF ㊁IGF -1及Hcy 与癫痫患者认知功能损害关系研究[J ].基因组学与应用生物学,2018,37(10):4547-4552.[13]㊀Xu H ,Nie B ,Liu L ,et al.Curcumin prevents brain damageand cognitive dysfunction during ischemic -reperfusion through the regulation of miR -7-5p [J ].Curr NeurovascRes ,2019,16(5):441-454.[14]㊀Li X ,Li G ,Jin Y ,et al.Long non -coding RNA maternallyexpressed 3(MEG3)regulates isoflurane -induced cogni-tive dysfunction by targeting miR -7-5p [J ].Toxicol Mech Methods ,2022,32(6):453-462.ʌ文章编号ɔ1006-6233(2024)04-0629-06三腔二囊管3种固定方法在食管胃底静脉曲张破裂出血患者压迫止血中的效果观察郭㊀蕾,㊀赵丽芳,㊀路㊀伟(空军军医大学第一附属医院消化急诊,㊀陕西㊀西安㊀710032)ʌ摘㊀要ɔ目的:探讨采用三腔二囊管3种固定方法在食管胃底静脉曲张破裂出血患者压迫止血中的应用疗效㊂方法:选取2021年1月至2022年10月我院收治的食管胃底静脉曲张破裂出血患者210例为研究对象,采用抽签法将患者完全随机分为传统固定组㊁鼻塞固定组㊁乒乓球固定组各70例,其中传统组采用传统法固定三腔二囊管,鼻塞固定组采用鼻塞法固定,乒乓球固定组采用乒乓球法固定㊂比较三组患者临床疗效(止血有效率㊁出血控制时间㊁输血量㊁再出血率)㊁并发症发生率(鼻黏膜破损㊁室息㊁胸闷㊁胸骨后疼痛㊁咽喉部损伤)及气囊破损率㊁置管成功率差异,记录治疗前及治疗后6个月时出血相关生化指标(血红蛋白㊁尿素氮㊁血小板数量)㊁凝血功能指标(纤维蛋白原含量㊁凝血酶原时间㊁凝血活酶时间)差异㊂结果:止血有效率㊁置管成功率组间比较结果均为乒乓球固定组(97.44%㊁100%)>鼻塞固定组(94.87%㊁97.14%)>传统固定组(87.50%㊁78.57%),且差异均具有统计学意义(P <0.05);三组患者临床疗效指标(出血控制时间㊁输血量)依次为乒乓球固定组<鼻塞固定组<传统固定组,且组间比较差异均具有统计学意义(P <0.05);与治疗前相比,三组患者治疗后6个月血红蛋白含量㊁血小板数量均明显升高(P <0.05),且升高幅度依次为乒乓球固定组>鼻塞固定组>传统固定组;尿素氮浓度明显降低(P <0.05),且降低幅度依次为乒乓球固定组>鼻塞固定组>传统固定组;三组患者再出血率㊁气囊破损率㊁凝血功能指标均无显著差异(P >0.05)㊂结论:与鼻塞固定法及传统固定法相比,乒乓球固定㊃926㊃ʌ基金项目ɔ陕西省自然科学基础研究计划项目,(编号:2018JQ8047)ʌ通讯作者ɔ路㊀伟。

沉默信息调节因子5在心血管疾病中的作用研究进展

网络出版时间：２０２４－０３－０９１８：４９：２４　网络出版地址：ｈｔｔｐｓ：／／ｌｉｎｋ．ｃｎｋｉ．ｎｅｔ／ｕｒｌｉｄ／３４．１０８６．ｒ．２０２４０３０６．１７２３．００４沉默信息调节因子５在心血管疾病中的作用研究进展于达云，许锦文，凌　霜（上海中医药大学交叉科学研究院，上海　２０１２０３）ｄｏｉ：１０．１２３６０／ＣＰＢ２０２２０６０５３文献标识码：Ａ文章编号：１００１－１９７８（２０２４）０３－０４０５－０５中国图书分类号：Ｒ０５；Ｒ３２９２４；Ｒ３４９１；Ｒ３６４５；Ｒ５４摘要：心血管疾病（ｃａｒｄｉｏｖａｓｃｕａｒｄｉｓｅａｓｅｓ，ＣＶＤｓ）的死亡率位居首位，严重威胁人类健康。

沉默信息调节因子５（ｓｉｌｅｎｔｉｎｆｏｒｍａｔｉｏｎｒｅｇｕｌａｔｏｒ５，ＳＩＲＴ５）作为一个新型的去酰基化修饰酶，广泛分布于心肌细胞、血管平滑肌细胞、内皮细胞等心血管细胞中，可通过脱乙酰化、脱琥珀酰化和脱丙二酰化而在多种ＣＶＤｓ中发挥重要作用。

该文将从能量代谢、调控炎症反应和氧化应激等方面讨论ＳＩＲＴ５的病理生理学机制，以及ＳＩＲＴ５在心肌梗塞、心肌肥大、心律失常、动脉粥样硬化和心收稿日期：２０２３－１０－１６，修回日期：２０２４－０１－０４基金项目：国家自然科学基金面上项目（Ｎｏ８１９７３５１１）作者简介：于达云（１９９８－），女，硕士生，研究方向：心血管中药药理学，Ｅｍａｉｌ：ｙｕｄａｙｕｎ２０１６＠１６３．ｃｏｍ；凌　霜（１９７９－），女，博士，副研究员，硕士生导师，研究方向：心血管中药药理学，通信作者，Ｅｍａｉｌ：ｓａｒａｈ＿ｌｉｎｇ＠１２６．ｃｏｍ力衰竭等ＣＶＤｓ中所扮演的角色，并且总结了目前ＳＩＲＴ５相关抑制剂和激动剂的研究进展，从而为靶向ＳＩＲＴ５提供防治ＣＶＤｓ的策略。

关键词：心血管疾病；沉默信息调节因子５；琥珀酰化；线粒体；能量代谢；炎症开放科学（资源服务）标识码（ＯＳＩＤ）：心血管疾病（ｃａｒｄｉｏｖａｓｃｕａｒｄｉｓｅａｓｅｓ，ＣＶＤｓ）是一系列涉及心脏、血管等循环系统的疾病，具有高患病率、高致残率、高复发率和高病死率的特点。

初级纤毛相关基因KIF3A通过Hedgehog信号通路影响口腔鳞状细胞癌增殖研究

延边大学医学学报　２０２２年３月　第４５卷　第１期与疾病报告２０２０概要［Ｊ］．中国循环杂志，２０２１，３６（６）：５２１－５４５．［２］　ＭＥＤＩＮＡ－ＬＥＹＴＥ　ＤＪ，ＺＥＰＥＤＡ－ＧＡＲＣíＡ　Ｏ，ＤＯＭíＮＧ－ＵＥＺＰéＲＥＺ　Ｍ，ｅｔ　ａｌ．．Ｅｎｄｏｔｈｅｌｉａｌ　ｄｙｓｆｕｎｃｔｉｏｎ，ｉｎｆｌａｍ－ｍａｔｉｏｎ　ａｎｄ　ｃｏｒｏｎａｒｙ　ａｒｔｅｒｙ　ｄｉｓｅａｓｅ：ｐｏｔｅｎｔｉａｌ　ｂｉｏｍａｒｋ－ｅｒｓ　ａｎｄ　ｐｒｏｍｉｓｉｎｇ　ｔｈｅｒａｐｅｕｔｉｃａｌ　ａｐｐｒｏａｃｈｅｓ［Ｊ］．Ｉｎｔ　ＪＭｏｌ　Ｓｃｉ，２０２１，２２（８）：３８５０．［３］　ＨＵＡＮＧ　Ｃ，ＨＵＡＮＧ　ＷＨ，ＷＡＮＧ　Ｒ，ｅｔ　ａｌ．．Ｕｌｉｎａｓｔａｔｉｎｉｎｈｉｂｉｔｓ　ｔｈｅ　ｐｒｏｌｉｆｅｒａｔｉｏｎ，ｉｎｖａｓｉｏｎ　ａｎｄ　ｐｈｅｎｏｔｙｐｉｃｓｗｉｔｃｈｉｎｇ　ｏｆ　ＰＤＧＦ－ＢＢ－ｉｎｄｕｃｅｄ　ＶＳＭＣｓ　ｖｉａ　Ａｋｔ／ｅＮＯＳ／ＮＯ／ｃＧＭＰ　ｓｉｇｎａｌｉｎｇ　ｐａｔｈｗａｙ［Ｊ］．Ｄｒｕｇ　Ｄｅｓ　Ｄｅｖｅｌ　Ｔｈｅｒ，２０２０，１４：５５０５－５５１４．［４］　赵高峰．延边地区朝鲜族ＳＭＡＲＣＡ４基因ＳＮＰｒｓ１１２２６０８与冠心病相关性研究［Ｄ］．延吉：延边大学，２０２０．［５］　ＩＭ　ＰＫ，ＭＩＬＬＷＯＯＤ　ＩＹ，ＫＡＲＴＳＯＮＡＫＩ　Ｃ，ｅｔ　ａｌ．．Ａｌ－ｃｏｈｏｌ　ｄｒｉｎｋｉｎｇ　ａｎｄ　ｒｉｓｋｓ　ｏｆ　ｔｏｔａｌ　ａｎｄ　ｓｉｔｅ－ｓｐｅｃｉｆｉｃ　ｃａｎｃ－ｅｒｓ　ｉｎ　Ｃｈｉｎａ：Ａ　１０－ｙｅａｒ　ｐｒｏｓｐｅｃｔｉｖｅ　ｓｔｕｄｙ　ｏｆ　０．５ｍｉｌｌｉｏｎａｄｕｌｔｓ［Ｊ］．Ｉｎｔ　Ｊ　Ｃａｎｃｅｒ，２０２１，１４９（３）：５２２－５３４．［６］　ＲＯＳＣＨ　ＰＪ．Ｃｏｕｌｄ　ｔｈｅ　ｂｅｎｅｆｉｔｓ　ｏｆ　ｄｒｉｎｋｉｎｇ　ａｌｃｏｈｏｌ　ｏｕｔ－ｗｅｉｇｈ　ａｌｌ　ｉｔｓ　ｒｉｓｋｓ［Ｊ］．Ｓｔｒｅｓｓ　Ｈｅａｌｔｈ，２０１３．［７］　ＷＡＬＬＥＲＡＴＨ　Ｔ，ＰＯＬＥＯ　Ｄ，ＬＩ　Ｈ，ｅｔ　ａｌ．．Ｒｅｄ　ｗｉｎｅ　ｉｎ－ｃｒｅａｓｅｓ　ｔｈｅ　ｅｘｐｒｅｓｓｉｏｎ　ｏｆ　ｈｕｍａｎ　ｅｎｄｏｔｈｅｌｉａｌ　ｎｉｔｒｉｃ　ｏｘｉｄｅｓｙｎｔｈａｓｅ：ａ　ｍｅｃｈａｎｉｓｍ　ｔｈａｔ　ｍａｙ　ｃｏｎｔｒｉｂｕｔｅ　ｔｏ　ｉｔｓ　ｂｅｎｅ－ｆｉｃｉａｌ　ｃａｒｄｉｏｖａｓｃｕｌａｒ　ｅｆｆｅｃｔｓ［Ｊ］．ＪＡＣＣ，２００３，４１（３）：４７１－４７８．［８］　ＺＨＡＯ　Ｆ，ＪＩ　Ｚ，ＣＨＩ　Ｊ，ｅｔ　ａｌ．．Ｅｆｆｅｃｔｓ　ｏｆ　Ｃｈｉｎｅｓｅ　ｙｅｌｌｏｗｗｉｎｅ　ｏｎ　ｎｉｔｒｉｃ　ｏｘｉｄｅ　ｓｙｎｔｈａｓｅ　ａｎｄ　ｉｎｔｅｒｃｅｌｌｕｌａｒ　ａｄｈｅｓｉｏｎｍｏｌｅｃｕｌｅ－１ｅｘｐｒｅｓｓｉｏｎｓ　ｉｎ　ｒａｔ　ｖａｓｃｕｌａｒ　ｅｎｄｏｔｈｅｌｉａｌ　ｃｅｌｌｓ［Ｊ］．Ａｃｔａ　Ｃａｒｄｉｏｌｏｇｉｃａ，２０１６，７１（１）：２７－３４．［９］　金春花，刘文博，关宏锏，等．一氧化氮合酶各种亚型的信号通路对心力衰竭的作用机制与治疗的研究进展［Ｊ］．中国循环杂志，２０１７，３２（８）：８３０－８３２．［１０］ＷＡＧＮＥＲ　ＭＣ，ＹＥＬＩＧＡＲ　ＳＭ，ＬＯＵ　ＡＢ，ｅｔ　ａｌ．．ＰＰＡＲγｌｉｇａｎｄｓ　ｒｅｇｕｌａｔｅ　ＮＡＤＰＨ　ｏｘｉｄａｓｅ，ｅＮＯＳ，ａｎｄ　ｂａｒｒｉｅｒｆｕｎｃｔｉｏｎ　ｉｎ　ｔｈｅ　ｌｕｎｇ　ｆｏｌｌｏｗｉｎｇ　ｃｈｒｏｎｉｃ　ａｌｃｏｈｏｌ　ｉｎｇｅｓｔｉｏｎ［Ｊ］．Ａｌｃｏｈｏｌ　Ｃｌｉｎ　Ｅｘｐ　Ｒｅｓ，２０１２，３６（２）：１９７－２０６．［１１］ＴＩＲＡＰＥＬＬＩ　ＣＲ，ＬＥＯＮＥ　ＡＦ，ＹＯＧＩ　Ａ，ｅｔ　ａｌ．．Ｅｔｈａｎｏｌｃｏｎｓｕｍｐｔｉｏｎ　ｉｎｃｒｅａｓｅｓ　ｂｌｏｏｄ　ｐｒｅｓｓｕｒｅ　ａｎｄ　ａｌｔｅｒｓ　ｔｈｅ　ｒｅ－ｓｐｏｎｓｉｖｅｎｅｓｓ　ｏｆ　ｔｈｅ　ｍｅｓｅｎｔｅｒｉｃ　ｖａｓｃｕｌａｔｕｒｅ　ｉｎ　ｒａｔｓ［Ｊ］．Ｊ　Ｐｈａｒｍ　Ｐｈａｒｍａｃｏｌ，２００８，６０（３）：３３１－３４１．［１２］ＣＨＥＮ　ＳＳ，ＷＡＮＧ　ＺＹ，ＺＨＯＵ　Ｈ，ｅｔ　ａｌ．．Ｉｃａｒｉｉｎ　ｒｅ－ｄｕｃｅｓ　ｈｉｇｈ　ｇｌｕｃｏｓｅ－ｉｎｄｕｃｅｄ　ｅｎｄｏｔｈｅｌｉａｌ　ｐｒｏｇｅｎｉｔｏｒ　ｃｅｌｌｄｙｓｆｕｎｃｔｉｏｎ　ｖｉａ　ｉｎｈｉｂｉｔｉｎｇ　ｔｈｅ　ｐ３８／ＣＲＥＢ　ｐａｔｈｗａｙ　ａｎｄａｃｔｉｖａｔｉｎｇ　ｔｈｅ　Ａｋｔ／ｅＮＯＳ／ＮＯ　ｐａｔｈｗａｙ［Ｊ］．Ｅｘｐ　ＴｈｅｒＭｅｄ，２０１９，１８（６）：４７７４－４７８０．■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■［基金项目］　国家自然科学基金项目（编号：８１５６０１７９）．［收稿日期］　２０２２－０１－０３［作者简介］　黄银珠（１９９５—），女，硕士，研究方向为口腔颌面外科．［通信作者］　金成日，Ｅｍａｉｌ：ｊｃｒ１１０５＠１６３．ｃｏｍ．初级纤毛相关基因ＫＩＦ３Ａ通过Ｈｅｄｇｅｈｏｇ信号通路影响口腔鳞状细胞癌增殖研究黄银珠，李京旭，金成日（延边大学附属医院口腔科，吉林延吉１３３０００）［摘要］　［目的］探讨初级纤毛相关基因ＫＩＦ３Ａ通过Ｈｅｄｇｅｈｏｇ信号通路对口腔鳞状细胞癌增殖能力产生的影响．［方法］选择人舌鳞癌Ｔｃａ８１１３细胞株作为研究对象，分为ＫＩＦ３Ａ－ｓｉＲＮＡ转染组和对照组．采用蛋白印迹实验和实时荧光定量实验检测各组ＫＩＦ３Ａ、Ｓｈｈ、Ｇｌｉ１蛋白及ｍＲＮＡ的相对表达水平；利用ＭＴＴ细胞增殖实验观察Ｔｃａ８１１３细胞增殖能力的变化．［结果］蛋白印迹实验结果显示，与对照组比较，ＫＩＦ３Ａ－ｓｉＲＮＡ转染组ＫＩＦ３Ａ、Ｓｈｈ、Ｇｌｉ１蛋白表达水平均明显下调（Ｐ＜０．０５）；实时荧光定量实验结果显示，与对照组比较，ＫＩＦ３Ａ－ｓｉＲＮＡ转染组ＫＩＦ３Ａ、Ｓｈｈ、Ｇｌｉ１ｍＲＮＡ表达水平均明显下调（Ｐ＜０．０１）；ＭＴＴ细胞增殖实验结果显示，细胞培养２４ｈ时两组细胞增殖间差异无统计学意义（Ｐ＞０．０５），细胞培养４８、７２ｈ时ＫＩＦ３Ａ－ｓｉＲＮＡ转染组增殖率较对照组明显降低（Ｐ＜０．０１）．［结论］沉默初级纤毛相关基因ＫＩＦ３Ａ可能通过调控Ｈｅｄｇｅｈｏｇ信号通路抑制口腔鳞癌的增殖能力．［关键词］　口腔鳞状细胞癌；初级纤毛；ＫＩＦ３Ａ；Ｈｅｄｇｅｈｏｇ信号通路ＤＯＩ：１０．１６０６８／ｊ．１０００－１８２４．２０２２．０１．００２［中图分类号］　Ｒ　７３９．８［文献标志码］　Ａ［文章编号］　１０００－１８２４（２０２２）０１－０００７－０４·７·■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■Ｊｏｕｒｎａｌ　ｏｆ　Ｍｅｄｉｃａｌ　Ｓｃｉｅｎｃｅ　Ｙａｎｂｉａｎ　Ｕｎｉｖｅｒｓｉｔｙ　Ｍａｒ．２０２２　Ｖｏｌ．４５　Ｎｏ．１Ｅｆｆｅｃｔｓ　ｏｆ　ｐｒｉｍａｒｙ　ｃｉｌｉｕｍ－ａｓｓｏｃｉａｔｅｄ　ｇｅｎｅ　ＫＩＦ３Ａｏｎ　ｏｒａｌ　ｓｑｕａｍｏｕｓ　ｃｅｌｌｃａｒｃｉｎｏｍａ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｂｙ　Ｈｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ　ｐａｔｈｗａｙＨＵＡＮＧ　Ｙｉｎｚｈｕ，ＬＩ　Ｊｉｎｇｘｕ，ＪＩＮ　Ｃｈｅｎｇｒｉ（Ｄｅｐａｒｔｍｅｎｔ　ｏｆ　Ｓｔｏｍａｔｏｌｏｇｙ，Ａｆｆｉｌｉａｔｅｄ　Ｈｏｓｐｉｔａｌ　ｏｆ　Ｙａｎｂｉａｎ　Ｕｎｉｖｅｒｓｉｔｙ，Ｙａｎｊｉ　１３３０００，Ｊｉｌｉｎ，Ｃｈｉｎａ）ＡＢＳＴＲＡＣＴ：ＯＢＪＥＣＴＩＶＥＴｏ　ｅｘｐｌｏｒｅ　ｔｈｅ　ｅｆｆｅｃｔ　ｏｆ　ｐｒｉｍａｒｙ　ｃｉｌｉｕｍ－ａｓｓｏｃｉａｔｅｄ　ｇｅｎｅ　ＫＩＦ３Ａｏｎ　ｏｒａｌ　ｓｑｕａｍｏｕｓｃｅｌｌ　ｃａｒｃｉｎｏｍａ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｔｈｒｏｕｇｈ　Ｈｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ　ｐａｔｈｗａｙ．ＭＥＴＨＯＤＳ　Ｈｕｍａｎ　ｔｏｎｇｕｅ　ｓｑｕａｍｏｕｓ　ｃｅｌｌｃａｒｃｉｎｏｍａ　Ｔｃａ８１１３ｃｅｌｌ　ｌｉｎｅ　ｗａｓ　ｓｅｌｅｃｔｅｄ　ａｎｄ　ｄｉｖｉｄｅｄ　ｉｎｔｏ　ＫＩＦ３Ａ－ｓｉＲＮＡ　ｔｒａｎｓｆｅｃｔｉｏｎ　ｇｒｏｕｐ　ａｎｄ　ｃｏｎｔｒｏｌｇｒｏｕｐ．Ｗｅｓｔｅｒｎ　ｂｌｏｔ　ａｎｄ　ｒｅａｌ－ｔｉｍｅ　ｑＰＣＲ　ｗｅｒｅ　ｕｓｅｄ　ｔｏ　ｄｅｔｅｃｔ　ｔｈｅ　ｒｅｌａｔｉｖｅ　ｅｘｐｒｅｓｓｉｏｎ　ｌｅｖｅｌｓ　ｏｆ　ＫＩＦ３Ａ，Ｓｈｈａｎｄ　Ｇｌｉ１ｐｒｏｔｅｉｎｓ　ａｎｄ　ｍＲＮＡ　ｉｎ　ｅａｃｈ　ｇｒｏｕｐ．ＭＴＴ　ｃｅｌｌ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ａｓｓａｙ　ｗａｓ　ｕｓｅｄ　ｔｏ　ｏｂｓｅｒｖｅ　ｔｈｅｐｒｏｌｉｆｅｒａｔｉｏｎ　ｏｆ　Ｔｃａ８１１３ｃｅｌｌｓ．ＲＥＳＵＬＴＳ　Ｗｅｓｔｅｒｎ　ｂｌｏｔ　ｒｅｓｕｌｔｓ　ｓｈｏｗｅｄ　ｔｈａｔ　ｃｏｍｐａｒｅｄ　ｗｉｔｈ　ｔｈｅ　ｃｏｎｔｒｏｌ　ｇｒｏｕｐ，ｔｈｅ　ｐｒｏｔｅｉｎ　ｅｘｐｒｅｓｓｉｏｎ　ｌｅｖｅｌｓ　ｏｆ　ＫＩＦ３Ａ，Ｓｈｈ　ａｎｄ　Ｇｌｉ１ｉｎ　ｔｈｅ　ＫＩＦ３Ａ－ＳｉＲＮＡ　ｔｒａｎｓｆｅｃｔｉｏｎ　ｇｒｏｕｐ　ｗｅｒｅｓｉｇｎｉｆｉｃａｎｔｌｙ　ｄｏｗｎ－ｒｅｇｕｌａｔｅｄ（Ｐ＜０．０５）．Ｒｅａｌ－ｔｉｍｅ　ｑＰＣＲ　ｒｅｓｕｌｔｓ　ｓｈｏｗｅｄ　ｔｈａｔ　ｃｏｍｐａｒｅｄ　ｗｉｔｈ　ｔｈｅ　ｃｏｎｔｒｏｌｇｒｏｕｐ，ｔｈｅ　ｍＲＮＡ　ｅｘｐｒｅｓｓｉｏｎ　ｌｅｖｅｌｓ　ｏｆ　ＫＩＦ３Ａ，Ｓｈｈ　ａｎｄ　Ｇｌｉ１ｉｎ　ｔｈｅ　ＫＩＦ３Ａ－ＳｉＲＮＡ　ｔｒａｎｓｆｅｃｔｉｏｎ　ｇｒｏｕｐ　ｗｅｒｅｓｉｇｎｉｆｉｃａｎｔｌｙ　ｄｏｗｎ－ｒｅｇｕｌａｔｅｄ（Ｐ＜０．０１）．ＭＴＴ　ｃｅｌｌ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ａｓｓａｙ　ｓｈｏｗｅｄ　ｔｈａｔ　ｔｈｅｒｅ　ｗａｓ　ｎｏ　ｓｉｇｎｉｆｉｃａｎｔｄｉｆｆｅｒｅｎｃｅ　ｉｎ　ｃｅｌｌ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｂｅｔｗｅｅｎ　ｔｈｅ　ｔｗｏ　ｇｒｏｕｐｓ　ａｆｔｅｒ　２４ｈｃｅｌｌ　ｃｕｌｔｕｒｅ（Ｐ＞０．０５）．Ｔｈｅ　ｐｒｏｌｉｆｅｒａｔｉｏｎｒａｔｅ　ｏｆ　ｔｈｅ　ＫＩＦ３Ａ－ＳｉＲＮＡ　ｔｒａｎｓｆｅｃｔｉｏｎ　ｇｒｏｕｐ　ｗａｓ　ｓｉｇｎｉｆｉｃａｎｔｌｙ　ｌｏｗｅｒ　ｔｈａｎ　ｔｈａｔ　ｏｆ　ｔｈｅ　ｃｏｎｔｒｏｌ　ｇｒｏｕｐ　ａｆｔｅｒ　ｃｅｌｌｃｕｌｔｕｒｅ　ｆｏｒ　４８ａｎｄ　７２ｈ（Ｐ＜０．０１）．ＣＯＮＣＬＵＳＩＯＮＳｉｌｅｎｃｉｎｇ　ｐｒｉｍａｒｙ　ｃｉｌｉｕｍ－ａｓｓｏｃｉａｔｅｄ　ｇｅｎｅ　ＫＩＦ３Ａｍａｙｉｎｈｉｂｉｔ　ｔｈｅ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｏｆ　ｏｒａｌ　ｓｑｕａｍｏｕｓ　ｃｅｌｌ　ｃａｒｃｉｎｏｍａ　ｂｙ　ｒｅｇｕｌａｔｉｎｇ　Ｈｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ　ｐａｔｈｗａｙ．Ｋｅｙｗｏｒｄｓ：ｏｒａｌ　ｓｑｕａｍｏｕｓ　ｃｅｌｌ　ｃａｒｃｉｎｏｍａ；ｐｒｉｍａｒｙ　ｃｉｌｉａ；ＫＩＦ３Ａ；Ｈｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ　ｐａｔｈｗａｙ口腔癌人数约占全世界罹患恶性肿瘤人数的３％，其中鳞状细胞癌比例超过９０％．２００５年，ＷＨＯ将口腔鳞状细胞癌（ＯＳＣＣ）定义为一种具有不同分化程度和侵袭性的肿瘤，早期可出现广泛的淋巴结转移，好发于４０～７０岁的烟酒爱好者［１］．口腔癌患者５年生存率约为５０％［２］，患者术后预后差和５年生存率低的主要原因为患者未及时就诊、淋巴结转移及复发［３］．控制ＯＳＣＣ的侵袭转移能力对预后至关重要，认为可偿试从分子水平出发找出更好的治疗方法［４］．初生纤毛属于一种细胞表面的细胞器，在脊椎动物发育和人类遗传疾病中发挥着重要作用．纤毛对发育信号的响应是必需的，而越来越多的证据表明初级纤毛是专属于Ｈｅｄｇｅｈｏｇ信号传导的［５］，而Ｈｅｄｇｅｈｏｇ信号传导对肿瘤的发生发展起着重要作用．研究［６］结果显示，多种肿瘤组织中均存在Ｈｅｄｇｅｈｏｇ信号的异常激活，如肺小细胞癌、胰腺癌、前列腺癌、乳腺癌、恶性胶质瘤、髓母细胞瘤及多发性骨髓瘤等．ＫＩＦ３Ａ属于初级纤毛内运输系统，具有维持和发挥初级纤毛正常功能的作用［７］．本研究探讨了初级纤毛相关基因ＫＩＦ３Ａ通过Ｈｅｄｇｅｈｏｇ信号通路对ＯＳＣＣ增殖能力产生的影响．１　材料与方法１．１　细胞　人舌鳞癌细胞株Ｔｃａ８１１３购自美国菌种保藏中心（ＡＴＣＣ），置于延边大学附属医院中心实验室封存．１．２细胞培养与转染　取Ｔｃａ８１１３细胞置于温度３７℃、二氧化碳体积分数５％、饱和湿度９５％的恒温孵育箱中，用含１０％（质量分数）ＦＢＳ、青链霉素的ＤＭＥＭ高糖型培养基传代培养．在转染前１ｄ，将Ｔｃａ８１１３细胞分为对照组和ＫＩＦ３Ａ－ｓｉＲＮＡ转染组后接种至６孔板中，待生长至８０％时进行细胞转染．ＫＩＦ３Ａ－ｓｉＲＮＡ转染组转染ＫＩＦ３Ａ－ｓｉＲＮＡ，对照组未进行转染．转染操作按照ＬｉｐｏｆｅｃｔａｍｉｎｅＴＭ２０００Ｒｅ－ａｇｅｎ转染说明书进行．ＫＩＦ３Ａ－ｓｉＲＮＡ序列（５′－３′）：ＵＡＡ　ＧＧＡ　ＡＵＧ　ＣＵＧ　ＡＡＧ　ＡＡＧ　ＡＣＧ　ＡＧＵ　Ｃ．１．３　蛋白印迹实验　转染２４ｈ后分别收集两组细胞，并加入ＲＩＰＡ与ＰＭＳＦ混合（１００:１）冰上裂解细胞，提取细胞总蛋白，采用ＢＣＡ试剂盒测定总蛋白水平．取各组标本行８％（质量分数）十二烷基硫酸钠聚丙烯酰胺凝胶（ＳＤＳ－ＰＡＧＥ）电泳，转膜，室温下用５％（质量分数）脱脂奶粉封闭２ｈ．ＴＢＳＴ洗涤３·８·■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■延边大学医学学报　２０２２年３月　第４５卷　第１期次，每次１０ｍｉｎ，加入兔抗人Ｋｉｆ３ａ一抗（１:１　０００）、兔抗人Ｓｈｈ一抗（１:２　０００）、兔抗人Ｇｌｉ１（１:１　０００）及兔抗人ＧＡＰＤＨ（１:１　０００）后置于湿盒中，于４℃冰箱中孵育过夜；次日ＴＢＳＴ洗涤３次，每次１０ｍｉｎ，分别加入山羊抗兔ＩｇＧ二抗（１:１０　０００）后置于湿盒中室温孵育２ｈ；ＴＢＳＴ洗涤３次，每次１０ｍｉｎ，加入增强型化学发光剂ＥＣＬ显影．以ＧＡＰＤＨ为内参，采用Ｉｍａｇｉｎｅ　Ｊ软件分析蛋白条带灰度值．１．４　实时荧光定量实验　转染２４ｈ后分别收集两组细胞，采用ＴＲＮｚｏｌ法提取细胞中的总ＲＮＡ，反转录为ｃＤＮＡ，按照ＲＴ－ＰＣＲ试剂盒说明书以ｃＤＮＡ为模板进行扩增．测定循环阈值（Ｃｔ），采用２－△△Ｃｔ法进行统计．实验重复３次．引物序列，ＫＩＦ３Ａ－正向：５′－ＡＧＡ　ＧＣＧ　ＴＣＡ　ＡＣＧ　ＡＧＧ　ＴＧＴ　ＴＴ－３′；ＫＩＦ３Ａ－反向：５′－ＴＡＴ　ＴＧＡ　ＴＣＧ　ＧＣＡ　ＴＣＴ　ＴＧＧ　ＣＣＣ－３′；Ｓｈｈ－正向：５′－ＣＴＣ　ＧＣＴ　ＧＣＴ　ＧＧＴ　ＡＴＧ　ＣＴＣ　Ｇ－３′；Ｓｈｈ－反向：５′－ＡＴＣ　ＧＣＴ　ＣＧＧ　ＡＧＴ　ＴＴＣ　ＴＧＧＡＧＡ－３′；Ｇｌｉ１－正向：５′－ＡＧＣ　ＧＴＧ　ＡＧＣ　ＣＴＧ　ＡＡＴＣＴＧ　ＴＧ－３′；Ｇｌｉ１－反向：５′－ＣＡＧ　ＣＡＴ　ＧＴＡ　ＣＴＧＧＧＣ　ＴＴＴ　ＧＡＡ－３′；ＧＡＰＤＨ－正向：５′－ＧＧＡ　ＧＣＧＡＧＡ　ＴＣＣ　ＣＴＣ　ＣＡＡ　ＡＡＴ－３′；ＧＡＰＤＨ－反向：５′－ＧＧＣ　ＴＧＴ　ＴＧＴＣＡＴＡＣＴ　ＴＣＴＣＡＴＧＧ－３′．１．５　ＭＴＴ细胞增殖实验　转染２４ｈ后分别收集两组细胞，以５　０００个／孔接种于９６孔板中，每组设置５个复孔．培养２４、４８、７２ｈ后弃去旧培养液，每孔加入１００μＬ　ＭＴＴ（５　ｇ／Ｌ），于３７℃、５％（体积分数）二氧化碳、９５％饱和湿度的恒温孵育箱中培养４　ｈ，弃去上清液终止反应，每孔加入１００μＬ　ＤＭＳＯ，置于振荡器３　ｍｉｎ溶解结晶后在酶标仪波长４９０　ｎｍ处测定各孔的光密度（ＯＤ）值，计算相对细胞增殖率．相对细胞增殖率（％）＝（实验组ＯＤ值／对照组ＯＤ值）×１００％．１．６　统计学分析　采用Ｇｒａｐｈｐａｄ　Ｐｒｉｓｍ　７．０软件进行数据分析．计量数据以均数±标准偏差（ｘ±ｓ）表示，进行两个样本均数的ｔ检验，以Ｐ＜０．０５为差异有统计学意义．２　结果２．１　沉默ＫＩＦ３Ａ基因后对Ｔｃａ８１１３细胞Ｈｅｄｇｅｈｏｇ信号通路中Ｓｈｈ、Ｇｌｉ１蛋白表达水平的影响　与对照组比较，ＫＩＦ３Ａ－ｓｉＲＮＡ转染组２４ｈ时ＫＩＦ３Ａ、Ｓｈｈ及Ｇｌｉ１蛋白表达水平均明显降低，差异均具有统计学意义（Ｐ＜０．０５），见Ｆｉｇｕｒｅ　１．ｃｏｍｐａｒｅｄ　ｗｉｔｈ　ｃｏｎｔｒｏｌ，＊Ｐ＜０．０５．Ｆｉｇｕｒｅ　１　Ｔｈｅ　ｐｒｏｔｅｉｎ　ｅｘｐｒｅｓｓｉｏｎ　ｌｅｖｅｌｓ　ｏｆ　ＫＩＦ３Ａ，Ｓｈｈａｎｄ　Ｇｌｉ１ｄｅｃｒｅａｓｅｄ　ｓｉｇｎｉｆｉｃａｎｔｌｙ　ａｆｔｅｒｔｒａｎｓｆｅｃｔｉｏｎ　ｏｆ　Ｔｃａ８１１３ｃｅｌｌｓ２．２　沉默ＫＩＦ３Ａ基因后对Ｔｃａ８１１３细胞中Ｈｅｄｇｅｈｏｇ信号通路Ｓｈｈ、Ｇｌｉ１ｍＲＮＡ表达水平的影响　与对照组比较，ＫＩＦ３Ａ－ｓｉＲＮＡ转染２４ｈ组ＫＩＦ３Ａ、Ｓｈｈ及Ｇｌｉ１ｍＲＮＡ表达水平均显著降低，差异均具有统计学意义（Ｐ＜０．０１），见Ｆｉｇｕｒｅ　２．ｃｏｍｐａｒｅｄ　ｗｉｔｈ　ｃｏｎｔｒｏｌ，＊＊Ｐ＜０．０１．Ｆｉｇｕｒｅ　２　Ｔｈｅ　ｍＲＮＡ　ｅｘｐｒｅｓｓｉｏｎ　ｌｅｖｅｌｓ　ｏｆ　ＫＩＦ３Ａ，Ｓｈｈａｎｄ　Ｇｌｉ１ｄｅｃｒｅａｓｅｄ　ｓｉｇｎｉｆｉｃａｎｔｌｙ　ａｆｔｅｒｔｒａｎｓｆｅｃｔｉｏｎ　ｏｆ　Ｔｃａ８１１３ｃｅｌｌｓ２．３　沉默ＫＩＦ３Ａ基因后对Ｔｃａ８１１３细胞增殖能力的影响　ＭＴＴ检测结果显示，细胞培养２４ｈ时ＫＩＦ３Ａ－ｓｉＲＮＡ转染组与对照组细胞增殖间差异无统计学意义（Ｐ＞０．０５）；细胞培养４８、７２ｈ时，ＫＩＦ３Ａ－ｓｉＲＮＡ转染组与对照组比较增殖显著受到抑制（Ｐ＜０．０１），见Ｆｉｇｕｒｅ　３．·９·■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■Ｊｏｕｒｎａｌ　ｏｆ　Ｍｅｄｉｃａｌ　Ｓｃｉｅｎｃｅ　Ｙａｎｂｉａｎ　Ｕｎｉｖｅｒｓｉｔｙ　Ｍａｒ．２０２２　Ｖｏｌ．４５　Ｎｏ．１ｃｏｍｐａｒｅｄ　ｗｉｔｈ　ｃｏｎｔｒｏｌ，＊＊Ｐ＜０．０１．Ｆｉｇｕｒｅ　３　Ｔｈｅ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｒａｔｅ　ｏｆ　Ｔｃａ８１１３ｃｅｌｌｓ　ａｆｔｅｒｔｒａｎｓｆｅｃｔｉｏｎ３　讨论初级纤毛为多数分化型细胞表面上的感觉附属物，具有化学感觉和机械感觉功能，在细胞周期控制中具有重要的作用．以往的研究认为初级纤毛在快速增殖的细胞上是找不到的，如癌症细胞，但ＫＯＷＡＬ等［８］通过一种初级纤毛标志物Ａｒｌ１３ｂ在ＨｅＬａ和ＭＧ６３细胞中发现了初级纤毛．未在ＯＳＣＣ中查看是否存在初级纤毛是本研究的不足之处．ＫＩＦ３Ａ属于初级纤毛内运输系统，在维持和发挥初级纤毛功能中起重要作用．ＨＯＡＮＧ－ＭＩＮＨ等［９］的研究结果显示，沉默ＫＩＦ３Ａ基因表达后可降低胶质母细胞瘤中初级纤毛的数量，最终促进肿瘤的发生．玄云泽等［１０］首次在ＯＳＣＣ中发现有初级纤毛，后来有学者亦在口腔白斑与ＯＳＣＣ组织中发现了初级纤毛，ＥＧＦＲ－Ａｕｒｏｒａ　Ａ异常信号被激活后，口腔黏膜癌变过程中原发性纤毛逐渐减少［１１］．本研究结果显示，利用ｓｉＲＮＡ技术沉默ＫＩＦ３Ａ基因后ｍＲＮＡ和蛋白表达水平明显降低，提示初级纤毛的检出率降低．Ｈｅｄｇｅｈｏｇ信号通路对胚胎发育及肿瘤的发生发展具有重要意义．研究［１２－１３］结果显示，Ｈｅｄｇｅｈｏｇ信号通路的异常激活与皮肤、乳腺、肺及消化道等多种恶性肿瘤的发生、增殖密切相关．程志芬等［１２］利用Ｈｅｄｇｅｈｏｇ信号通路特异性抑制剂环巴胺处理Ｔｃａ８１１３细胞株后，Ｓｍｏｏｔｈｅｎｅｄ的ｍＲＮＡ和蛋白表达明显减少，且细胞增殖受到抑制．ＫＵＲＯＤＡ等［１４］的研究结果显示，给予Ｈｅｄｇｅｈｏｇ信号通路抑制剂可抑制肿瘤前沿血管内皮细胞的增殖、迁移，进而抑制肿瘤的增殖和迁移．在小细胞肺癌中，沉默ＫＩＦ３Ａ后初级纤毛明显减少，且Ｓｈｈ信号通路介导的Ｇｌｉ１ｍＲＮＡ表达水平明显降低［１５］，此结果与本研究结果相符．研究［１６］结果显示，Ｈｅｄｇｅｈｏｇ信号通路转录因子Ｇｌｉ１的激活是恶性肿瘤的一个不良预后因素，利用环巴胺阻断Ｈｅｄｇｅｈｏｇ信号通路可抑制Ｇｌｉ１激活，并增强头颈部鳞癌对放疗的敏感性，提示Ｈｅｄｇｅｈｏｇ信号通路影响头颈部鳞癌的放疗预后．总之，本研究揭示了初级纤毛相关基因ＫＩＦ３Ａ在人舌鳞癌Ｔｃａ８１１３细胞Ｈｅｄｇｅｈｏｇ信号通路中的作用，初步解释了沉默ＫＩＦ３Ａ后可能通过抑制Ｈｅｄｇｅｈｏｇ信号通路影响ＯＳＣＣ细胞的增殖能力，为ＯＳＣＣ患者的靶向治疗提供了理论依据．［参　考　文　献］［１］　ＤＩ　ＰＡＲＤＯ　ＢＪ，ＢＲＯＮＳＯＮ　ＮＷ，ＤＩＧＧＳ　ＢＳ，ｅｔ　ａｌ．．Ｔｈｅｇｌｏｂａｌ　ｂｕｒｄｅｎ　ｏｆ　ｅｓｏｐｈａｇｅａｌ　ｃａｎｃｅｒ：ａ　ｄｉｓａｂｉｌｉｔｙ－ａｄｊｕｓｔｅｄｌｉｆｅ－ｙｅａｒ　ａｐｐｒｏａｃｈ［Ｊ］．Ｗｏｒｌｄ　Ｊ　Ｓｕｒｇ，２０１６，４０（２）：３９５－４０１．［２］　ＢＲＯＣＫＬＥＨＵＲＳＴ　ＰＲ，ＢＡＫＥＲ　ＳＲ，ＳＰＥＩＧＨＴ　ＰＭ．Ｏｒａｌｃａｎｃｅｒ　ｓｃｒｅｅｎｉｎｇ：ｗｈａｔ　ｈａｖｅ　ｗｅ　ｌｅａｒｎｔ　ａｎｄ　ｗｈａｔ　ｉｓ　ｔｈｅｒｅｓｔｉｌｌ　ｔｏ　ａｃｈｉｅｖｅ［Ｊ］．Ｆｕｔｕｒｅ　Ｏｎｃｏｌ，２０１０，６（２）：２９９－３０４．［３］　ＢＡＶＬＥ　ＲＭ，ＶＥＮＵＧＯＰＡＬ　Ｒ，ＫＯＮＤＡ　Ｐ，ｅｔ　ａｌ．．Ｍｏ－ｌｅｃｕｌａｒ　ｃｌａｓｓｉｆｉｃａｔｉｏｎ　ｏｆ　ｏｒａｌ　ｓｑｕａｍｏｕｓ　ｃｅｌｌ　ｃａｒｃｉｎｏｍａ［Ｊ］．ＪＣＤＲ，２０１６，１０（９）：Ｚ１８－Ｚ２１．［４］　陈新，徐文华，周健，等．口腔鳞状细胞癌现状［Ｊ］．口腔医学，２０１７，３７（５）：４６２－４６５．［５］　ＧＯＥＴＺ　ＳＣ，ＡＮＤＥＲＳＯＮ　ＫＶ．Ｔｈｅ　ｐｒｉｍａｒｙ　ｃｉｌｉｕｍ：ａ　ｓｉｇｎａｌｌｉｎｇ　ｃｅｎｔｒｅ　ｄｕｒｉｎｇ　ｖｅｒｔｅｂｒａｔｅ　ｄｅｖｅｌｏｐｍｅｎｔ［Ｊ］．Ｎａｔ　Ｒｅｖ　Ｇｅｎｅ，２０１０，１１（５）：３３１－３４４．［６］　那玉岩，刘万林．纤毛相关疾病：细胞学机制及转化应用进展［Ｊ］．中国组织工程研究，２０１６，２０（２４）：３６４２－３６４８．［７］　ＮＩＥＷＩＡＤＯＭＳＫＩ　Ｐ，ＮＩＥＤＺＩółＫＡ　ＳＭ，ＭＡＲＫＩＥＷＩＣＺŁ，ｅｔ　ａｌ．．Ｇｌｉ　ｐｒｏｔｅｉｎｓ：ｒｅｇｕｌａｔｉｏｎ　ｉｎ　ｄｅｖｅｌｏｐｍｅｎｔ　ａｎｄｃａｎｃｅｒ［Ｊ］．Ｃｅｌｌｓ，２０１９，８（２）：１４７．［８］　ＫＯＷＡＬ　ＴＪ，ＦＡＬＫ　ＭＭ．Ｐｒｉｍａｒｙ　ｃｉｌｉａ　ｆｏｕｎｄ　ｏｎ　ＨｅＬａａｎｄ　ｏｔｈｅｒ　ｃａｎｃｅｒ　ｃｅｌｌｓ［Ｊ］．Ｃｅｌｌ　Ｂｉｏｌ　Ｉｎｔ，２０１５，３９（１１）：１３４１－１３４７．［９］　ＨＯＡＮＧ－ＭＩＮＨ　ＬＢ，ＤＥＬＥＹＲＯＬＬＥ　ＬＰ，ＳＩＥＢＺＥＨ－ＮＲＵＢＬ　Ｄ，ｅｔ　ａｌ．．Ｄｉｓｒｕｐｔｉｏｎ　ｏｆ　ＫＩＦ３Ａｉｎ　ｐａｔｉｅｎｔ－ｄｅ－ｒｉｖｅｄ　ｇｌｉｏｂｌａｓｔｏｍａ　ｃｅｌｌｓ：ｅｆｆｅｃｔｓ　ｏｎ　ｃｉｌｉｏｇｅｎｅｓｉｓ，ｈｅｄｇｅ－ｈｏｇ　ｓｅｎｓｉｔｉｖｉｔｙ，ａｎｄ　ｔｕｍｏｒｉｇｅｎｅｓｉｓ［Ｊ］．Ｏｎｃｏｔａｒｇｅｔ，２０１６，７（６）：７０２９－７０４３．［１０］玄云泽，李书进，金成日，等．初级纤毛相关基因ＫＩＦ３Ａ通过调控ＥＭＴ过程在口腔鳞状细胞癌中的作用机制研究［Ｊ］．重庆医学，２０２０，４９（１）：７－１２．［１１］ＹＩＮ　Ｆ，ＣＨＥＮ　Ｑ，ＳＨＩ　Ｙ，ｅｔ　ａｌ．．Ａｃｔｉｖａｔｉｏｎ　ｏｆ　ＥＧＦＲ－Ａｕ－ｒｏｒａ　Ａ　ｉｎｄｕｃｅｓ　ｌｏｓｓ　ｏｆ　ｐｒｉｍａｒｙ　ｃｉｌｉａ　ｉｎ　ｏｒａｌ　ｓｑｕａｍｏｕｓ　ｃｅｌｌｃａｒｃｉｎｏｍａ［Ｊ］．Ｏｒａｌ　Ｄｉｓ，２０２２，２８（３）：６２１－６３０．［１２］程志芬，崔演，玄延花．Ｐｔｃｈ和Ｓｍｏ在舌鳞状细胞癌Ｔｃａ８１１３细胞中的表达及其意义［Ｊ］．口腔医学研究，２０１５，３１（５）：４３３－４３６．［１３］ＫＯＮＳＴＡＮＴＩＮＯＵ　Ｄ，ＢＥＲＴＡＵＸ－ＳＫＥＩＲＩＫ　Ｎ，ＺＡＶＲＯＳＹ．Ｈｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ　ｉｎ　ｔｈｅ　ｓｔｏｍａｃｈ［Ｊ］．Ｃｕｒｒ　ＯｐｉｎＰｈａｒｍａｃｏｌ，２０１６，３１：７６－８２．［１４］ＫＵＲＯＤＡ　Ｈ，ＫＵＲＩＯ　Ｎ，ＳＨＩＭＯ　Ｔ，ｅｔ　ａｌ．．Ｏｒａｌ　ｓｑｕａｍｏｕｓｃｅｌｌ　ｃａｒｃｉｎｏｍａ－ｄｅｒｉｖｅｄ　ｓｏｎｉｃ　ｈｅｄｇｅｈｏｇ　ｐｒｏｍｏｔｅｓ　ａｎｇｉｏｇｅｎｅ－ｓｉｓ［Ｊ］．Ａｎｔｉｃａｎｃｅｒ　Ｒｅｓ，２０１７，３７（１２）：６７３１－６７３７．［１５］ＣＯＣＨＲＡＮＥ　ＣＲ，ＶＡＧＨＪＩＡＮＩ　Ｖ，ＳＺＣＺＥＰＮＹ　Ａ，ｅｔ　ａｌ．．Ｔｒｐ５３ａｎｄ　Ｒｂ１ｒｅｇｕｌａｔｅ　ａｕｔｏｐｈａｇｙ　ａｎｄ　ｌｉｇａｎｄ－ｄｅｐｅｎｄｅｎｔＨｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ［Ｊ］．Ｊ　Ｃｌｉｎ　Ｉｎｖｅｓｔ，２０２０，１３０（８）：４００６－４０１８．［１６］ＧＡＮ　ＧＮ，ＥＡＧＬＥＳ　Ｊ，ＫＥＹＳＡＲ　ＳＢ，ｅｔ　ａｌ．．Ｈｅｄｇｅｈｏｇｓｉｇｎａｌｉｎｇ　ｄｒｉｖｅｓ　ｒａｄｉｏｒｅｓｉｓｔａｎｃｅ　ａｎｄ　ｓｔｒｏｍａ－ｄｒｉｖｅｎｔｕｍｏｒ　ｒｅｐｏｐｕｌａｔｉｏｎ　ｉｎ　ｈｅａｄ　ａｎｄ　ｎｅｃｋ　ｓｑｕａｍｏｕｓ　ｃａｎｃｅｒｓ［Ｊ］．Ｃａｎｃｅｒ　Ｒｅｓ，２０１４，７４（２３）：７０２４－７０３６．■·０１·■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■。

α7nAChR的抗炎及神经保护作用研究进展

α7nAChR的抗炎及神经保护作用研究进展谢佳丽;刘超明;李良东;黄志华【摘要】烟碱型乙酰胆碱受体(Nicotinic acetylcholine receptors, nAChRs)是配体门控离子通道蛋白,广泛分布于中枢和外周神经系统,在神经元和非神经元细胞中表达,参与各种生理反应.其亚型α7nAChR的抗炎及神经保护作用日益受到关注,被认为是一个潜在的治疗神经系统疾病和炎症的作用靶点.本文总结了α7nAChR在神经系统及非神经系统的分布和表达,在免疫反应中的作用(胆碱能抗炎通路),以及神经保护作用,并指出小胶质细胞α7nAChR可作为抗炎通路治疗靶点.【期刊名称】《赣南医学院学报》【年(卷),期】2017(037)003【总页数】6页(P471-476)【关键词】α7nAChR;炎症;神经保护作用;研究进展【作者】谢佳丽;刘超明;李良东;黄志华【作者单位】赣南医学院,江西赣州 341000;赣南医学院,江西赣州 341000;赣南医学院基础医学院,江西赣州 341000;赣南医学院基础医学院,江西赣州 341000【正文语种】中文【中图分类】R364.5炎症是一个重要的宿主反应，对机体稳态发挥十分重要的作用。

在中枢神经系统(central nervous system, CNS)中，小胶质细胞类似固有免疫系统的巨噬细胞，虽然小胶质细胞可清除细胞碎片，进行组织愈合和修理，但其慢性激活参与了神经退行性疾病的发病过程[1]。

烟碱型乙酰胆碱受体(Nicotinic acetylcholine receptors, nAChRs)属于“半胱氨酸环” 配体门控离子通道超家族，在记忆、学习、运动、关注和焦虑等多个生理进程中起着重要的作用。

其中α7nAChR参与多种生理功能，如神经保护、突触可塑性和神经元的存活等，且被认为在各种各样的精神及神经疾病中扮演重要角色。

在外周及中枢，高表达α7nAChR的免疫细胞起抑制炎症的作用[2]，被认为是一个突出的抗炎治疗靶点。

AhR信号通路与HIF-1信号通路的相互作用

M OLECULAR AND C ELLULAR B IOLOGY,July2009,p.3465–3477Vol.29,No.13 0270-7306/09/$08.00ϩ0doi:10.1128/MCB.00206-09Copyright©2009,American Society for Microbiology.All Rights Reserved.The Active Form of Human Aryl Hydrocarbon Receptor(AHR)Repressor Lacks Exon8,and Its Pro185and Ala185Variants Repressboth AHR and Hypoxia-Inducible Factorᰔ†Sibel I.Karchner,1Matthew J.Jenny,1Ann M.Tarrant,1Brad R.Evans,1,2Hyo Jin Kang,3Insoo Bae,3David H.Sherr,4and Mark E.Hahn1*Biology Department,Woods Hole Oceanographic Institution,Woods Hole,Massachusetts025431;Biology Department, Boston University,Boston,Massachusetts022152;Department of Oncology,Georgetown University Medical Center, Washington,DC200073;and Department of Environmental Health,Boston University School ofPublic Health,Boston,Massachusetts021184Received16February2009/Accepted10April2009The aryl hydrocarbon receptor(AHR)repressor(AHRR)inhibits AHR-mediated transcription and has beenassociated with reproductive dysfunction and tumorigenesis in humans.Previous studies have characterizedthe repressor function of AHRRs from mice andﬁsh,but the human AHRR ortholog(AHRR715)appeared tobe nonfunctional in vitro.Here,we report a novel human AHRR cDNA(AHRR⌬8)that lacks exon8ofAHRR715.AHRR⌬8was the predominant AHRR form expressed in human tissues and cell lines.AHRR⌬8effectively repressed AHR-dependent transactivation,whereas AHRR715was much less active.Similarly,AHRR⌬8,but not AHRR715,formed a complex with AHR nuclear translocator(ARNT).Repression of AHR byAHRR⌬8was not relieved by overexpression of ARNT or AHR coactivators,suggesting that competition for these cofactors is not the mechanism of repression.AHRR⌬8interacted weakly with AHR but did not inhibit its nuclear translocation.In a survey of transcription factor speciﬁcity,AHRR⌬8did not repress the nuclear receptor pregnane X receptor or estrogen receptor␣but did repress hypoxia-inducible factor(HIF)-dependent signaling.AHRR⌬8-Pro185and-Ala185variants,which have been linked to human reproductive disorders,both were capable of repressing AHR or HIF.Together,these results identify AHRR⌬8as the active form of human AHRR and reveal novel aspects of its function and speciﬁcity as a repressor.The aryl hydrocarbon receptor(AHR)repressor(AHRR)is a basic-helix-loop-helix/Per-AHR nuclear translocator(ARNT)-Sim(bHLH-PAS)protein discovered because of its similarity to the AHR,a ligand-activated transcription factor involved in the response to synthetic aromatic hydrocarbons(48).The AHR and AHRR form a negative regulatory loop that is evolutionarily conserved in vertebrates(32);expression of AHRR is regulated by the AHR,and AHRR acts as a transcriptional repressor of AHR function(1,32,48).Like the AHR,AHRR can dimerize with the ARNT,and the AHRR-ARNT complex can bind to AHR-responsive enhancer elements(AHREs).Repression oc-curs through competition between AHR and AHRR for binding to AHREs(14,48)as well as through additional mechanisms that do not involve competition for ARNT and are independent of AHRE binding by AHRR(14).The biological and toxicological functions of AHRR are not well understood,but recentﬁndings suggest that AHRR is involved in human reproductive physiology and in the regula-tion of cell growth(reviewed in references20and22).A human AHRR(hAHRR)Ala185Pro polymorphism has been associated with altered reproductive development and infertil-ity in men(16,46,59,64)and endometriosis in women(19,35,62,65),but the functional properties of the polymorphic vari-ants have never been assessed.AHRR overexpression inhibits the growth of human tumor cells in culture(30,56,68).Con-versely,knockdown of AHRR expression enhances cell growth and confers resistance to apoptosis;consistent with this,the AHRR gene has been found to be silenced by hypermethylation in a variety of human cancers(71).Based on these and other ﬁndings,the AHRR has been proposed to function as a tumor suppressor gene(22,71).In order to assess the functions of AHRR and its polymor-phic variants and their relationship to human disease,it is important to understand the nature of the transcripts and proteins encoded by the AHRR gene,as well as their expres-sion in human tissues and cell lines.An hAHRR cDNA iden-tiﬁed in a large-scale screen of cDNAs from brain(50)encodes a protein of715amino acids(aa)(referred to here as AHRR715).The human AHRR gene encoding this protein has been reported to contain12exons,theﬁrst of which is non-coding(8,16).Our initial functional analysis of this protein suggested that,unlike AHRRs from mouse,frog,andﬁsh(15,32,48,70),human AHRR715was not an effective repressor of AHR function in vitro.In phylogenetic analyses involving amino acid sequence alignments of multiple vertebrateAHRRs,we identiﬁed an18-aa segment of AHRR715that was absent from all other AHRRs.We therefore hypothesized the existence of an alternative hAHRR form lacking this segment and also hypothesized that this alternative form might exhibit characteristic repressor function.*Corresponding author.Mailing address:Biology Department,MS#32,Woods Hole Oceanographic Institution Woods Hole,MA 02543.Phone:(508)289-3242.Fax:(508)457-2134.E-mail:mhahn@.†Supplemental material for this article may be found at http://mcb/.ᰔPublished ahead of print on20April2009.3465Here,we report the identiﬁcation and cloning of a novel hAHRR cDNA that lacks exon8of the original AHRR clone. This new AHRR(AHRR⌬8)is the predominant form ex-pressed in multiple human tissues and human tumor cell lines. We compare the functions of the two AHRR splice variants and provide theﬁrst functional mechanistic assessment of the hAHRR Pro185Ala polymorphic variants that have been as-sociated with increased susceptibility to reproductive dysfunc-tion in human populations.We also show that competition for ARNT or AHR coactivators is not involved in the mechanism of AHR repression and that human AHRR⌬8(hAHRR⌬8)is capable of repressing hypoxia-inducible factor(HIF)-depen-dent signaling.MATERIALS AND METHODSChemicals and cell lines.2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD)was obtained from Ultra Scientiﬁc(Hope,RI).Dimethyl sulfoxide(DMSO),clo-trimazole,cobalt chloride,and17␤-estradiol were obtained from Sigma-Aldrich (St.Louis,MO).COS-7cells and the human cell lines HepG2,HeLa,MCF-7, Hs578T,and MDA231were obtained from the American Type Culture Collec-tion(ATCC;Manassas,VA)and grown according to standard procedures.BP-1 cells were generously provided by J.Russo(Fox Chase Cancer Center,Phila-delphia,PA).Human cDNA panel screening.The expression of AHRR exon8in various human tissues was determined by amplifying a partial fragment of AHRR with primersﬂanking the exon8region.Primers hRR-F635(5Ј-AGTACTCGGCCT TCCTGACC-3Ј)and hRR-R816(5Ј-CGCCTTCTTCTTCTGTCCAA-3Ј)were used with5␮l of cDNAs from adult and fetal human cDNA panels(BD Biosciences,Mountain View,CA)in a25-␮l ampliﬁcation reaction mixture using AmpliTaqGold DNA polymerase(Applied Biosystems,Foster City,CA).The PCR conditions were as follows:94°C for10min,94°C for15s and60°C for30s for35cycles,and72°C for7min.The PCR products were resolved on15% Tris-borate EDTA gels.Screening of human cell lines for the Pro/Ala polymorphism and the presence of exon8.Total RNA was isolated from human cell lines MCF-7,Hs578T, MDA231,and BP-1as described earlier(68).Brieﬂy,cells were frozen and pulverized into aﬁne powder.Total cellular RNA was isolated using RNAzol as described by the manufacturer(Leedo Medical Laboratories,Houston,TX). RNA was quantiﬁed with a spectrophotometer at optical densities of260nm and 280nm.cDNA was synthesized from2␮g of total RNA using Omniscript reverse transcriptase(Qiagen,Valencia,CA).A partial fragment of AHRR was ampli-ﬁed from cDNA derived from each cell line using primers hRR-F494(5Ј-AGG ACTTCTGCCGGCAGCTCC-3Ј)and RRex8R(5Ј-CAGCTGCCAAGCCTGT GACC-3Ј)ﬂanking the region containing the Pro185Ala polymorphism and exon 8.The PCR products were cloned into the pGEM-T vector(Promega),and multiple clones were sequenced for each cell line.Generation of AHRR plasmid constructs.The pcDNAhAHRR(AHRR715) construct was prepared by subcloning the KIAA1234cDNA(clone fH08618;a gift from Takahiro Nagase,Kazusa DNA Research Institute,Chiba,Japan[50]) into pcDNA3.1,as we described earlier(32).Full-length hAHRR⌬8was ampli-ﬁed from testes cDNA(human cDNA panel;BD Biosciences,Mountain View, CA)using primers hRR-F39(5Ј-GATCATATGCCGAGGACGAT-3Ј)and hRR-R2227(5Ј-GAGCTTGGATGGTGGTCACT-3Ј)and Advantage DNA polymerase(BD Biosciences).The PCR conditions were as follows:94°C for1 min;94°C for5s,64°C for10s,and68°C for2.5min forﬁve cycles;94°C for5s, 62°C for10s,and68°C for2.5min forﬁve cycles;94°C for5s,60°C for10s,and 68°C for2.5min for25cycles;72°C for10min.The ampliﬁed product was cloned into the pGEM-T vector(Promega,Madison,WI)and sequenced.The insert was cut out of EcoRI and SpeI sites and transferred to the EcoRI and XbaI sites of the pcDNA3.1vector(Invitrogen,Carlsbad,CA).Other plasmid constructs.The pEF-hAHR and mouse AHR-yellowﬂuores-cent protein(YFP)fusion constructs were provided by Gary Perdew(Pennsyl-vania State University,University Park,PA).The plasmid pGudLuc6.1,which contains theﬁreﬂy luciferase reporter under the control of a mouse mammary tumor virus promoter regulated by four AHREs from the murine CYP1A1 promoter,was a gift from M.Denison(University of California,Davis,CA).Rat Cyp1a1-Luc and human XRE.1A1-Luc were obtained from R.Barouki(Univer-sity of Rene Descartes,France)and S.K.Kim(Seoul National University,South Korea),respectively.Expression constructs for human ARNT and the hypoxia-responsive luciferase reporter,HRE-luc(PL949[25]),were obtained from C. Bradﬁeld(University of Wisconsin,Madison,WI).The human pregnane X receptor(PXR)expression construct was provided by S.Kliewer(University of North Carolina at Chapel Hill),and the PXR reporter construct(XREM-tk-luc) was obtained from J.Moore(Molecular Discovery Research,GlaxoSmithKline, Research Triangle Park,NC).The human estrogen receptor␣(ER␣)construct and an estrogen-responsive luciferase reporter(3xERE-TATA-luc)were ob-tained from D.McDonnell(Duke University Medical Center,Durham,NC). Expression constructs for the receptor coactivators GRIP,CoCoA,and GAC63 were provided by M.Stallcup(University of Southern California,Los Angeles, CA).Src-1a and Src-1e constructs were obtained from E.Kalkhoven(University Medical Center Utrecht,The Netherlands)and M.Parker(Imperial College London,United Kingdom).The p300expression construct was from Upstate Biotechnologies(Lake Placid,NY).Transient transfections and luciferase assays.Transient transfections were performed as described earlier(14,32).Brieﬂy,transfections of DNA with Lipofectamine2000reagent(Invitrogen,Carlsbad,CA)were carried out in triplicate wells24h after plating.Approximately300ng of DNA was complexed with1␮l of Lipofectamine2000and then added to cells;the amount of DNA used for each expression construct is listed in theﬁgure legends.The total amount of DNA was kept constant by adding in empty vector.Five hours after transfection,cells were exposed to DMSO(0.5%),TCDD(10nMﬁnal concen-tration),clotrimazole(10␮Mﬁnal concentration),CoCl(150␮Mﬁnal concen-tration),or17␤-estradiol(10nMﬁnal concentration).(For transfections involv-ing17␤-estradiol and ER␣,cells were grown in phenol red-free medium with charcoal-stripped serum.)Renilla luciferase(pRL-TK or pGL4.74;Promega, Madison,WI)was used as the transfection control.Cells were lysed19h after dosing,and luminescence was measured using the dual luciferase assay kit (Promega)in a TD20/20luminometer(Turner Designs,Sunnyvale,CA).The ﬁnal values are expressed as a ratio of theﬁreﬂy luciferase units to the Renilla luciferase units.Experiments were repeated multiple times.AHRR antibody production and Western blots.Polyclonal antibodies to hAHRR(designed to recognize both forms)were raised in two rabbits(21st Century Biochemicals,Marlboro,MA)by coimmunizing the animals with two peptides corresponding to amino acid residues18to31(LQKQRPAVGAE KSN)and80to101(FQVVQEQSSRQPAAGAPSPGDS).To avoid cross-re-activity with AHR,the peptides were in regions of the AHRR protein exhibiting low sequence identity with the AHR(see Fig.S1in the supplemental material). Preimmune serum and serum from six bleeds were collected over the course of several weeks,and antibody titer was tested by Western blotting using hAHRR-transfected COS-7cell lysates;lysates from COS-7cells transfected with empty vector served as a control for speciﬁcity.COS-7cells were plated and transfected as described above.Twenty-four hours after transfection,cells were rinsed with phosphate-buffered saline and resuspended in2ϫsample treatment buffer.Cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electro-phoresis,and the gels were blotted onto nitrocellulose.The serum antibody titer for each bleed was tested by blotting with two dilutions(1:250and1:1,000).Two afﬁnity-puriﬁed polyclonal antibodies were isolated from serum(bleeds3thru6) from a single rabbit by separate afﬁnity puriﬁcation procedures using the two individual peptides.The afﬁnity-puriﬁed antibodies are designated PAb-RR-18-1 (against residues18to31)and PAb-RR-80-2(against residues80to101).The speciﬁcity of the afﬁnity-puriﬁed polyclonal antibodies was assessed by blotting against lysates from COS-7cells transfected with plasmids for hAHRR,human AHR,mouse AHRR,and killiﬁsh AHRR.All results reported here(Western blots and immunoprecipitations)were performed using PAb-RR-80-2. Expression of hAHRR protein in the transient-transfection assays was mea-sured by Western blotting with PAb-RR-80-2(3␮g/ml),followed by a goat anti-rabbit immunoglobulin G(IgG)Ϫhorseradish peroxidase(Upstate/Milli-pore,Billerica,MA)secondary antibody(1:5,000).The AHRR proteins were then visualized by enhanced chemiluminescence(ECL-Plus;GE Healthcare, Piscataway,NJ).Coimmunoprecipitation assay.The full-length AHRR715,AHRR⌬8,AHR, and ARNT proteins were synthesized by in vitro transcription and translation (TnT;Promega,Madison,WI)in the presence or absence of[35S]methionine (MP Biomedicals,Solon,OH).Five microliters of unlabeled protein was mixed with15␮l of radiolabeled protein and incubated at room temperature for2h. For mixtures containing AHR,TCDD(10nM)was added.The mixtures were adjusted to25mM HEPES,200mM NaCl,1.2mM EDTA,10%glycerol,and 0.1%Nonidet P-40,pH7.4,with protease inhibitors(immunoprecipitation buffer).After two rounds of preclearing with normal mouse IgG and protein G-agarose,5␮g of speciﬁc antibody or IgG was added and incubated for2h, followed by precipitation with protein G-agarose overnight.The beads were washed two times with IP buffer,boiled in sample treatment buffer,and subjected3466KARCHNER ET AL.M OL.C ELL.B IOL.to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on an8%poly-acrylamide gel.The gels were dried and visualized byﬂuorography.hAHRR antibody(PAb-RR-80-2)or normal rabbit IgG was used to precipitate AHRR complexes and nonspeciﬁc complexes,respectively.For ARNT complexes, monoclonal ARNT antibody(MA1-515;Afﬁnity BioReagents,Golden,CO)and normal mouse IgG were used.Subcellular localization of mouse AHR-YFP.COS-7cells were grown on coverslips in six-well plates.Cells were cotransfected with350ng of mouse AHR-YFP and350ng of human ARNT expression constructs,with or without 350ng of hAHRR⌬8construct,using Lipofectamine2000reagent(Invitrogen). Luciferase reporter pGudLuc6.1(300ng)and the transfection control pRL-TK (40ng)were also transfected.Cells were dosed with DMSO or TCDD(10nM ﬁnal concentration)5h after transfection.Twenty-four hours after transfection, cells were washed with1ϫphosphate-buffered saline andﬁxed in4%formalde-hyde.The coverslips were inverted onto slides and mounted with Vectashield hard-setting mounting medium(Vector Laboratories,Burlingame,CA).Cells were visualized using a Zeiss Axio Imager.Z1ﬂuorescence microscope,and Axiovision software was used to collect the images.To conﬁrm the effectiveness of AHRR⌬8as a repressor under the conditions of the assay,luciferase was measured in a plate of cells run in parallel.Nucleotide sequence accession numbers.The AHRR⌬8sequences have been deposited in GenBank,with accession numbers EU293605(mRNA)and ABX89616(protein).RESULTSIdentiﬁcation and characterization of the major form of hAHRR.In our earlier studies of the evolutionary conservation of AHRR in vertebrates(15,32),we noticed that the predicted 715-aa protein derived from the original hAHRR cDNA(AHRR715[50])included an18-aa segment that had no coun-terpart(homologous amino acids)in other AHRRs.A more recent phylogenetic analysis involving an alignment of all known(i.e.,veriﬁed)mammalian,amphibian,andﬁsh AHRR protein sequences conﬁrmed that this unique18-aa segment ispresent only in the human AHRR715(Fig.1A).This segmentis located in an otherwise conserved portion of the PAS region downstream of the PAS-A repeat(sometimes referred to as the“intervening region”[10]between PAS repeats)(see Fig. S1in the supplemental material);it is encoded by a single exon of54nucleotides in the human genome(Fig.1B),correspond-ing to exon8described by others(8,16).AHRR715,the715-aaprotein encoded by the cDNA containing this exon,functioned poorly as a repressor in transient-transfection assays in three different laboratories(unpublished results;see below).We therefore hypothesized that there might be an alternatively spliced AHRR transcript lacking this exon and that the protein encoded by this alternative transcript might have a repressor function like that of AHRRs from other species.Using primersﬂanking exon8,we used PCR to amplify this region of the AHRR transcript from human tissue cDNAs. The primary amplicon was128bp,the size predicted for a form lacking exon8,rather than the182bp predicted for the exon 8-containing transcript.Subsequently,we ampliﬁed,cloned, and sequenced a full-length cDNA of2,173bp with an open reading frame of2091bp encoding a predicted AHRR proteinof697aa.The new cDNA is identical to AHRR715(50),exceptthat it lacks the sequences corresponding to exon8and thus has been designated AHRR⌬8.Two polymorphic variants of AHRR⌬8,corresponding to the Ala185Pro single nucleotide polymorphism described earlier(65),were found among the sequenced clones.To assess the relative expression of AHRR715andAHRR⌬8,we performed PCR analysis on cDNA from human tissues with primersﬂanking exon8,designed to produce am-plicons of different sizes for the two AHRR variants.A survey of multiple adult and fetal tissues demonstrated that AHRR⌬8 is the predominant,and in most cases only,form of AHRR expressed(Fig.1C).We also examined the relative expression of AHRR715and AHRR⌬8and the presence of Ala185Pro polymorphic variants in several human tumor cell lines.As seen with the human tissues,AHRR⌬8was the predominant form of AHRR expressed in HeLa,HepG2,MCF-7,Hs578T, MDA231,and BP-1cells(see Fig.S2A and Table S1in thesupplemental material).Although AHRR715transcripts were not detected by using primersﬂanking exon8,use of a primerwithin exon8showed that AHRR715transcripts were ex-pressed at low levels in HeLa and HepG2cells(see Fig.S2B in the supplemental material).Sequencing of AHRR cDNA clones from each of the cell lines conﬁrmed AHRR⌬8as the predominant expressed form and showed that all of the cell lines except BP-1are heterozygous for the Ala185Pro poly-morphism(see Table S1in the supplemental material). AHRR⌬8and AHRR differ in repressor activity.To com-pare the functional properties of the original715-aa AHRRprotein(AHRR715)and AHRR⌬8,we performed transient-transfection assays in which we measured the ability of the AHRRs to inhibit the TCDD-inducible transactivation of re-porter gene construct pGudLuc6.1mediated by either trans-fected or endogenously expressed AHR.After transfectionwith the respective constructs,AHRR715and AHRR⌬8pro-teins were expressed at similar levels in COS-7cells,as as-sessed by Western blotting using an hAHRR antibody that recognizes both AHRR forms but does not recognize human AHR(Fig.2A and B).When transfected into COS-7cells with ARNT in the presence or absence of TCDD,neither AHRR form was able to activate transcription of pGudLuc6.1(see Fig. S3A in the supplemental material).Thus,hAHRRs lack a function as transcriptional activators,as observed previously for AHRRs from other species(15,32,48).To test the ability of AHRR715and AHRR⌬8to repress AHR-mediated signaling,each form was transfected into COS-7cells together with expression constructs for human AHR and ARNT and pGudLuc6.1.In the absence of cotrans-fected AHRR,AHR and ARNT caused transactivation of the luciferase reporter that was enhanced by TCDD(Fig.2C).Transfection of the AHRR715expression construct at50and 150ng/well caused little change in the AHR-dependent acti-vation of luciferase expression or its induction by TCDD.In contrast,AHRR⌬8at50or150ng/well completely repressed both constitutive(i.e.,exogenous ligand-independent)and TCDD-inducible reporter gene activity(Fig.2C).Similarly,AHRR⌬8,but not AHRR715,repressed transactivation by mouse AHR in COS-7cells(see Fig.S3B in the supplemental material).To evaluate the effect of the AHRRs on endog-enously expressed human AHR,AHRR715or AHRR⌬8was cotransfected with pGudLuc6.1into HepG2cells,which ex-press abundant AHR(13).As we saw with the transfectedAHRs in COS-7cells,AHRR715was ineffective as a repressor of the endogenous HepG2AHR,whereas AHRR⌬8reduced TCDD-inducible reporter gene activation by61%(Fig.2D).AHRR⌬8also was much more effective than AHRR715at repressing endogenous AHR in MCF-7cells cotransfected with two different reporter gene constructs(Cyp1a1-Luc andV OL.29,2009HUMAN AHRR⌬8AND VARIANTS REPRESS AHR AND HIF3467XRE.1A1-Luc)(Fig.2E;see also Fig.S3C in the supplemental material).(The lower percent repression in HepG2and MCF-7cells than in COS-7cells likely reﬂects the fact that endogenous AHR is expressed in all HepG2and MCF-7cells,whereas not all of the cells take up transiently transfected AHRR.)Together,these results demonstrate that the much greater ability of AHRR ⌬8than of AHRR 715to repress AHR transactivation is consistently observed for transfected and en-dogenously expressed AHRs from humans and mice,in three different cell lines,and with AHRE-containing reporter gene constructs derived from three different mammalian species.AHRR ⌬8and AHRR 715differ in ability to interact with ARNT.The 18-aa peptide encoded by exon 8in AHRR 715lies in the ϳ100-aa “intervening region”downstream of the PAS-A repeat.This region is highly conserved in AHR and AHRR proteins (Fig.1A;see also Fig.S1in the supplemental mate-rial),and in the AHR,it has been shown to be important for dimerization with ARNT (10,45).This region also appears to be important for dimerization of AHRR and ARNT,because deletion of this region in the zebraﬁsh AHRRa caused a dra-matic reduction in the ability of AHRRa to interact with ARNT (14).To determine whether the presence of the 18-aa peptide within the intervening region of AHRR 715affects its ability to dimerize with ARNT,we performed a coimmuno-precipitation experiment in which radiolabeled ARNT was in-cubated with in vitro-expressed AHRR ⌬8or AHRR 715and the complex was immunoprecipitated with afﬁnity-puriﬁed antibody against hAHRR,which recognizes both forms.AHRR ⌬8and AHRR 715were synthesized to similar levels by in vitro transcription and translation (Fig.3A).AHRR ⌬8FIG.1.Identiﬁcation of hAHRR splice variant AHRR ⌬8as the major variant expressed in human tissues.(A)Partial amino acid sequence alignment of AHRRs and AHRs from different species.The top sequence is human AHRR 715;immediately below it is the sequence of AHRR ⌬8.Abbreviations:Hs,human;Mm,mouse;Rn,rat;Xl,frog;Fh,killiﬁsh;Tr,Japanese puffer ﬁsh;Dr,zebraﬁsh;Mt,tomcod.For GenBank accession numbers,see the legend to Fig.S1in the supplemental material.(B)hAHRR gene structure.Translated exons are shown in black boxes.The ﬁrst exon and part of the second exon are untranslated.The additional exon (exon 8)is shown in gray.The position of the Pro185Ala polymorphism is marked by an arrow.(C)Survey of adult human tissues for the expression of AHRR 715and AHRR ⌬8transcripts.A partial AHRR cDNA fragment was ampliﬁed from adult and fetal human cDNAs,using primers ﬂanking exon 8(shown in gray in panel B).The presence of the exon would have produced a 182-bp PCR product (AHRR 715),as opposed to the 128-bp product (AHRR ⌬8)obtained in all tissues.3468KARCHNER ET AL.M OL .C ELL .B IOL .formed a complex with ARNT that could be speciﬁcally and strongly immunoprecipitated by the AHRR antibody (Fig.3B,lane 3versus lane 4;see also Fig.S4B in the supplemental material).In contrast,AHRR 715did not interact with ARNT (Fig.3B,lane 1versus lane 2;see also Fig.S4B in the supple-mental material).The results suggest that the presence of the 18-aa peptide encoded by exon 8disrupts ARNT dimerization,that hAHRR requires ARNT to repress AHR,and that the difference in the repressor function of AHRR ⌬8and AHRR 715reﬂects the inability of the latter protein to associate with ARNT.Functional comparison of AHRR ⌬8-Ala 185and AHRR ⌬8-Pro 185polymorphic variants.An Ala185Pro polymorphism in the hAHRR has been associated with human diseases in sev-eral studies (16,19,35,46,59,62,64,65),but the functional properties of the two variants have never been assessed.Both of these polymorphic variants were present in our pool of AHRR ⌬8clones.To compare their abilities to repress AHR transactivation,we performed transient-transfection assays in which we measured the abilities of AHRR ⌬8-Ala 185and AHRR ⌬8-Pro 185to repress AHR-and ARNT-dependent transactivation of pGudLuc6.1in COS-7cells.The AHRR ⌬8variants were expressed at similar levels in the transfected cells (Fig.4A).Both AHRR ⌬8-Ala 185and AHRR ⌬8-Pro 185were effective at repressing constitutive (exogenous ligand-indepen-dent)and TCDD-inducible expression of pGudLuc6.1.In ex-periments in which increasing amounts of AHRR ⌬8expres-sion constructs were transfected,the two variants were equally potent at repressing AHR-mediated transactivation of the re-porter gene (Fig.4B and C).We conclude that both AHRR ⌬8-Ala 185and AHRR ⌬8-Pro 185are fully functional as repressors of AHR and thus that the Ala185Pro polymorphism does not affect repression of AHR-mediated transcription.Mechanism of repression of AHR by AHRR ⌬8.Mimura et al.(48)showed that mouse AHRR could dimerize with ARNT and bind to AHREs and proposed that the mechanism of repression involved competition between AHR and AHRR for binding to ARNT and for binding to AHRE sequences.Our recent studies using the zebraﬁsh AHRRa provided evidence that competition for ARNT is not an important element of the mechanism of repression and that AHRE binding may con-tribute to the repression but is not required (14).To assess the role of competition for ARNT in the repression of human AHR by hAHRR ⌬8,we performed a series oftransient-trans-FIG.2.Repressor activity of hAHRR splice variants AHRR 715and AHRR ⌬8.(A)The hAHRR antibody does not recognize human AHR or AHRRs from other species.Cell lysates from COS-7cells transiently expressing the indicated constructs were blotted and probed with antibody PAb-RR-80-2raised against the hAHRR.(B)In transient-transfection assays in COS-7cells,AHRR 715and AHRR ⌬8are expressed at similar levels.Cell lysates were blotted and probed with the hAHRR antibody.Numbers at left of panels A and B are molecular masses in kilodaltons.(C)Repression of exogenously expressed AHR by AHRR 715and AHRR ⌬8in COS-7cells.COS-7cells were transfected with human AHR (5ng),human ARNT (25ng),and AHRR 715or AHRR ⌬8constructs (50or 150ng each),along with a luciferase reporter under the control of dioxin response elements (pGudLuc6.1)and a transfection control plasmid expressing Renilla luciferase (pRL-TK).Cells were dosed with DMSO or TCDD (10nM ﬁnal concentration),followed by a luciferase assay.The results shown are representative of seven independent experiments.(D and E)Repression of endogenous AHR in HepG2(D)and MCF-7(E)cells by AHRR 715and AHRR ⌬8.Cells were transfected with AHRR 715or AHRR ⌬8constructs (25and 100ng each),along with a luciferase reporter under the control of dioxin response elements (for HepG2,pGudLuc6.1;for MCF-7,Cyp1a1-Luc)and pRL-TK.Cells were dosed with DMSO or TCDD (10nM ﬁnal concentration),followed by a luciferase assay.The results shown in panels D and E are representative of two and three independent experiments,respectively.V OL .29,2009HUMAN AHRR ⌬8AND VARIANTS REPRESS AHR AND HIF 3469。

BBRC模板范文

Limited effect of recombinant human mannose-binding lectinon the infection of novel inﬂuenza A(H7N9)virus in vitroJinlei Guo a,1,Yang Cao b,1,Kun Qin a,Xiaopeng Zhao a,Donghong Wang a,Zi Li a,Li Xin a, Yuelong Shu a,Jianfang Zhou a,*a National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Key Laboratory for Medical Virology, National Health and Family Planning Commission,Beijing102206,PR Chinab Center of Growth,Metabolism and Aging,Key Laboratory of Bio-Resource and Eco-Environment,Ministry of Education,College of Life Sciences,Sichuan University,No.29Wangjiang Road,Chengdu610064,PR Chinaa r t i c l e i n f oArticle history:Received23December2014 Available online26January2015Keywords:Mannose-binding lectinInﬂuenza A virusInnate immunityH7N9a b s t r a c tMannose-binding lectin(MBL),a pattern-recognition molecule in serum,recognizes speciﬁc hexose sugars rich in mannose and N-acetylglucosamine on bacterium,yeasts,viruses as well as apoptotic cells. It has been well-identiﬁed that MBL has antiviral effects via binding to seasonal inﬂuenza H1and H3 subtype viruses.Inﬂuenza A(H7N9)virus,a novel reassortant virus to human population,possesses the surface hemagglutinin(HA)and neuraminidase(NA)genes from duck and wild-bird inﬂuenza viruses and internal genes from poultry H9N2viruses.As of Dec7th,2014,a total of467human infections and 183fatal cases have been identiﬁed.Here,recombinant human(rh)MBL was tested for its binding and effects on hemagglutination inhibition(HI)and NA activity inhibition(NAI)of avian H7N9,H9N2and human H3N2viruses.We discovered that rhMBL exhibited a strong binding to H7N9virus as human H3N2did at high virus titers.However,it performed a signiﬁcantly weaker HI activity effect on H7N9 comparing to those of H3N2and H9N2,even at a much higher concentration(3.67±0.33vs.0.026±0.001and0.083±0.02m g/mL,respectively).Similarly,minor NAI effect of rhMBL,even at up to 10m g/mL,was found on H7N9virus while it displayed signiﬁcant effects on both H3N2and H9N2at a lowest concentration of0.0807±0.009and0.0625m g/mL,respectively.The HI and NAI effects of rhMBL were calcium-dependent and mediated by lectin domain.Ourﬁndings suggest that MBL,the host innate molecule,has differential interference effects with human and avian inﬂuenza virus and limited antiviral effect against H7N9virus.©2015The Authors.Published by Elsevier Inc.This is an open access article under the CC BY-NC-NDlicense(/licenses/by-nc-nd/4.0/).1.IntroductionHost innate immunity plays a critical role in the early phase of infection.Thisﬁrst-line defense against pathogens is mediated by a variety of pattern-recognition molecules including collectins,toll-like receptors andﬁcolins as well as inﬂammatory cytokines and type I interferon or macrophages and natural killer cells.Mannose-binding lectin(MBL)is one of collectins circulating in the serum and synthesized by liver.It consists of collagenous domains and carbohydrate recognition domains(CRD).The CRDs recognize sugars including D-mannose,N-acetylmannosamine,N-acetylglu-cosamine and L-fucose on the surface of many pathogens in a calcium-dependent manner[1].Previous studies showed that MBL can bind to a range of clinically relevant microorganisms such as Staphylococcus aureus,Candida Albicans[2],HIV,SARS-CoV,Ebola virus,HSV,inﬂuenza virus[3e6].The binding of MBL to microor-ganisms is presumed to induce MBL conformational changes that allow the molecule to initiate viral neutralization or kill virus via opsonization or complement activation[7].Inﬂuenza A virus,a segmented single-stranded negative-sense RNA virus,belongs to orthomyxoviridae and is subtyped according to the antigenic properties of their envelope glycoproteins,HA and NA.Currently,16HA subtypes and9NA subtypes circulate in birds. Among them,only seasonal H1N1and H3N2viruses circulate in human population[8].Occasionally,some subtypes of avian*Corresponding author.National Institute for Viral Disease Control and Preven-tion,Chinese Center for Disease Control and Prevention,Key Laboratory for Medical Virology,National Health and Family Planning Commission,155Changbai Road, Beijing102206,PR China.Fax:þ8601063580764.E-mail address:jfz@(J.Zhou).1These authors contributed equally in thisstudy.Contents lists available at ScienceDirectBiochemical and Biophysical Research Communications jou rn al homepage:/locate/ybbrc/10.1016/j.bbrc.2015.01.0700006-291X/©2015The Authors.Published by Elsevier Inc.This is an open access article under the CC BY-NC-ND license(/licenses/by-nc-nd/4.0/).Biochemical and Biophysical Research Communications458(2015)77e81inﬂuenza A virus can jump into human and cause diseases with a range of clinical symptoms and outcomes,such as conjunctivitis, mild upper respiratory tract disease,as well as severe pneumonia and death[9e12].Viral HA and NA assist virus binding,entry and releasing during infection cycle.Their potential N-linked glycosyl-ation sites(NGS)can be glycosylated,which might allow their binding to host MBL.It has been found that the glycan at residue 165in H3N2HA was of high-mannose and MBL neutralized viral infectivity via it.Many lines of evidences have shown that the MBL plays an important role inﬁghting against seasonalﬂu[13e15]. However,little is known about the interactions between avian inﬂuenza virus and the innate molecules.Avian inﬂuenza H7N9 virus is novel to human population[16,17],which contains the surface HA and NA genes from duck and wild-bird inﬂuenza viruses and internal genes from poultry H9N2viruses.Unlike other H7 viruses that generally cause mild symptoms such as conjunctivitis or inﬂuenza-like illness(except one fatal case infected with H7N7 in Netherlands in2003),H7N9virus usually results in severe pneumonia or respiratory failure in human.Here,we examined the interactions of MBL with avian inﬂuenza virus H7N9,H9N2and human virus H3N2.Furthermore,we studied the molecule mech-anisms for them by structure modeling.2.Materials and methods2.1.VirusThe vaccine strain A/Anhui/1/2013(H7N9)(NIBRG-268)was ob-tained from National Institute for Biological Standards and Control (UK),namely H7N9Vac.The virus bears the HA and NA of A/Anhui/1/ 2013(H7N9)and internal genes of A/Puerto Rico/8/1934(PR8,H1N1); A/Brisbane/10/2007(H3N2)was named as H3N2WT in the study; H9N2virus,a reassortant bearing the HA,NA from A/Hongkong/ 33982/2009(H9N2)and internal genes of PR8,was named as H9N2RG.The reassortant H7N1AH1HAþPR8NA was with HA of A/ Anhui/1/2013and seven genes of PR8,which is rescued as previously reported[18].H7N9Vac,H3N2WT and H7N1AH1HAþPR8NA were propagated in9e11-day-old embryonated chicken eggs,H9N2RG was grown in Madin-Darby canine kidney(MDCK)cells(ATCC,USA) with Modiﬁed Eagle's Medium(invitrogen,USA)containing2m g/mL N-tosyl-L-phenylalanine chloromethyl ketone(TPCK)e treated trypsin(Sigma,USA).Virus stocks were puriﬁed by adsorption to and elution from turkey red blood cells(TRBCs)and stored atÀ80 Cuntil use[19].Virus titer was determined by titration in MDCK cells and the tissue culture infectious dose affecting50%of the cultures (TCID50)is calculated by the Reed e Muench formula[20].2.2.Detection of MBL binding to inﬂuenza virusRecombinant human MBL(rhMBL)was purchased from Sino Biological Inc(Beijing,China).Ninety-six-well plates were coated with2Â105TCID50inﬂuenza virus at a volume of100m l/well for overnight at4 C,then were blocked for1h with1%Bovine Serum Albumin(BSA,Roche,Switzerland)at37 C.Different concentra-tions of rhMBL(0,1,3,5,7m g/mL)were added and incubated for 1h at37 C.The virus-dose dependent binding assay was con-ducted as that wells were precoated with2Â102,2Â103,2Â104 and2Â105TCID50inﬂuenza viruses per well.Then3m g/mL rhMBL was added and incubated for1h at37 C.The binding was detected by the biotinylated human MBL pAb(0.2m g/mL)(R&D, USA),followed by streptavidin-horseradish peroxidase(HRP) (1:200)(R&D,USA)and tetramethylbenzidine substrate solution (BD,USA),the reaction was stopped by2M H2SO4and the Optical Density(OD)at450nm was measured by ELISA reader(Perkin-Elmer,USA).The wells coated with10m g/mL mannan from Saccharomyces cerevisiae(Sigma,USA)or coating buffer(Kirke-gaard&Perry Laboratories,USA)were used as positive control and negative control respectively.The test was performed in duplicates and in three independent experiments,absorbance from negative control was subtracted and results were normalized to positive control,data was expressed as a relative absorbance value using mean±SEM(%).2.3.Hemagglutination inhibition(HI)assaysHI assay was performed in V-bottom96-well plates as previ-ously described[20].Brieﬂy,25m L inﬂuenza virus(4HAU)was mixed with25m L rhMBL of different concentrations diluted in Hank's Balanced Salt Solution(HBSS)containing1.26mM Ca2þfor 1h at37 C,then50m L1%TRBC was added to the mixture and incubate at room temperature for30min.For HI reverse assay: rhMBL was diluted in HBSS containing10mM EDTA or10mg/mL mannan,then incubated with4HAU of inﬂuenza virus.The results were expressed as the minimum inhibitory concentration(MIC)of rhMBL that exhibited HI effect.2.4.Neuraminidase activity inhibition(NAI)assaysInﬂuenza virus NA activity was measured by ELISA in which peanut agglutinin conjugated with HRP was used to detect b-D-galactose-N-acetylglucosamine exposed after removal of sialic acid from fetuin[21].Appropriate amounts of virus in Dulbecco's1X PBS with CaCl2and MgCl2(Life Technologies,USA)were used to perform the NAI assays.Different concentrations of rhMBL were diluted in HBSS containing Ca2þand mixed with inﬂuenza virus in a total volume of100m L and preincubated at37 C for1h,and then transferred to wells precoated with fetuin(Sigma,USA)and incu-bated at37 C for4h,After washing,100m L of HRP-labeled peanut lectin(3m g/mL)was added and after1h at room temperature,the wells were washed and o-phenylenediamine dihydrochloride in citrate buffer was added,reaction was stopped by2M H2SO4,and the OD at492nm was measured.The wells only with virus were used as the positive control,the OD of wells with HBSS used as a negative control was subtracted.Results were expressed as relative NA activity(%)calculated as the OD of the tested wells with virus and rhMBL divided by the OD of the wells with only virus.Table1Distances from the potential N-linked glycosylation sites(NGS)to receptor binding domain or NA activity region(Å).NGS Protein Distances from the NGS to functional region(Å)H3N2WT H9N2RG H7N9Vac63HA27.3e e95HA e23.2e122HA26.9e e128HA e17.6e126HA24.3e e133HA16e e144HA18.9e e165HA24.1e e198HA e16.2e240HA e e37.3246HA22.2e e86NA29.429.430146NA20.821.228.9200NA18.118.418.4234NA29.429.5e329NA26.5e e402NA22.422.7ee:Denotes the absence of NGS in the corresponding virus.J.Guo et al./Biochemical and Biophysical Research Communications458(2015)77e81 782.5.Structure modelingThe HA and NA 3D structures were predicted by using the ho-mology modeling method of SWISS-MODEL [22].The modeling structures,corresponding templates and identities are shown in Supplementary Table S1.Potential NGS were identi ﬁed with the NetNGlyc 1.0server (http://www.cbs.dtu.dk/services/NetNGlyc/)using arti ﬁcial neural networks that examine the context of Asn-Xaa-Ser/Thr sequences.The predicted NGS include 86,146,200,234,329and 402of NA,79,105,138,141,142,149,160,181,206,249and 262of HA (H3N2numbering)(Table 1).The NGS at the modeling structures were highlighted in Fig.S1with Pymol (DeLano Scienti ﬁc).The trimer structure of MBL was assembled using MBL crystal structure (PDB code:1HUP)with PISA [23]which generate the oligomeric forms of protein according to the symmetry information.To quantify the distance between RBD/NA activity region and each NGS,we employed the average Euclidean distance of the center (C alpha atom)of every RBD/NA activity region and the center (C alpha atom)of NGS.2.6.Statistical analysisDifferences between groups were tested using non-parametric Kruskal e Wallis analysis of variance (ANOVA)for multiple com-parisons using IBM SPSS Statistics 20.0(IBM,USA)and p <0.05was considered statistically signi ﬁcant.3.Results3.1.Binding of rhMBL with seasonal and avian in ﬂuenza viruses The ELISA results showed that rhMBL bound both seasonal and avian in ﬂuenza viruses in vitro .Incubation with increasingconcentrations of rhMBL resulted in elevated levels of MBL bound to immobilized in ﬂuenza virus,reaching a plateau at 3m g/mL (Fig.1A).Then,3m g/mL rhMBL was used to determine the af ﬁnity to increasing amounts of virus,revealing the virus-dose depen-dent binding feature (Fig.1B).High binding of rhMBL to the hu-man H3N2virus might be due to a relatively more NGS in its HA and NA (Table S2),in addition to the glycans attached at the site of 165as previously reported [14].Beyond our expectation,H7N9virus exhibited stronger binding than H9N2virus,reaching a comparable level as human H3N2at higher titers of 2Â104and 2Â105,although fewer NGS was observed in avian H7N9than avian H9N2(Table S2).The differential binding of rhMBL to avian virus might result from the types of oligosaccharide attached and further investigations are needed.The ﬁnding demonstrated here implied a possible antiviral effect initiated by rhMBL at early stage of novel avian virus infection since naïve host lacked the pre-existed cross-reactive or speci ﬁc anti-HA or anti-NA antibodies.3.2.Weak HI interference with H7N9virus by rhMBLInitially,we discovered that the lowest concentration for rhMBL (m g/mL,mean ±SEM)to abolish virus-mediated TRBC agglutination was 1.42±0.239,0.083±0.02,0.026±0.005for H7N9vac,H9N2RG and H3N2WT (Table 2),respectively.The HI effect could be reversed in the presence of 5mM EDTA or 5mg/mL mannan,which indicates that HI was calcium-dependent and mannan-inhibitable.We then used a H7N1reassortant obtained HA from H7N9and other seven segments from PR8to exclude a possible hemadsorption by N9reported elsewhere [24].The MIC against the H7N1was 3.67±0.33m g/mL (Table 2),which was remarkably higher than those against H3N2or H9N2virus,indicating a weaker HI effect of rhMBL onH7.Fig.1.Binding of rhMBL to puri ﬁed in ﬂuenza A virus.A.The dose-dependent binding of rhMBL to in ﬂuenza virus.Wells were precoated with 2Â105TCID 50in ﬂuenza virus H7N9Vac (C ),H9N2RG (-),H3N2WT (:),1%BSA (;).B.The bindings of rhMBL to in ﬂuenza virus increase with viral titer.Wells were precoated with 2Â102,2Â103,2Â104,2Â105TCID 50in ﬂuenza viruses H7N9Vac (C ),H9N2RG (-)and H3N2WT (:),3m g/mL rhMBL was used to detect the binding.Levels of rhMBL bound to immobilized in ﬂuenza virus were detected by ELISA,as described in Materials and methods.The absorbance from negative control was subtracted and the results were normalized to positive control,the mannan.Data are expressed as mean ±SEM (%)from three independent experiments.Table 2HI titer of rhMBL against in ﬂuenza A viruses.Concentrations inhibiting hemagglutination of IAV by rhMBL (m g/mL)H3N2WTH9N2RG H7N9Vac H7N1AH1HA þPR8NAMBL 0.026±0.0050.083±0.02 1.42±0.239 3.67±0.33þEDTA a >0.5ND ND ND þmannan b>0.5NDNDNDResults are expressed as mean ±SEM of three independent experiments.ND:not determined.H3N2WT:A/Brisbane/10/2007(H3N2)wild-type;H9N2RG:virus with the HA,NA from A/Hongkong/33982/2009(H9N2)and internal genes from A/Puerto Rico/8/1934(H1N1);H7N9Vac:virus with the HA,NA from A/Anhui/1/2013(H7N9)and internal genes from A/Puerto Rico/8/1934(H1N1);H7N1AH1HA þPR8NA :virus with HA from A/Anhui/1/2013(H7N9)and other seven genes from A/Puerto Rico/8/1934(H1N1).aThe HI test was performed in the presence of 5mM EDTA and rhMBL of different concentrations.The maxim concentration of MBL for testing is 0.5m g/mL.bThe HI test was performed in the presence of 5mg/mL mannan and rhMBL of different concentrations.The maxim concentration of MBL for testing is 0.5m g/mL.J.Guo et al./Biochemical and Biophysical Research Communications 458(2015)77e 81793.3.NAI of H3N2and H9N2by rhMBL not H7N9virusAs shown in Fig.2A,NA activity of H3N2was inhibited by rhMBL in a dose-dependent manner,and the IC 50was 0.0807±0.009m g/mL.Similarly,the rhMBL,at a concentration of 0.0625m g/mL,could inhibit the NA activity of H9N2(Fig.2B).Nevertheless,the relative NA activity of H7N9remained above 50%in the presence of increasing concentration of rhMBL even at up to 10m g/mL.By contrast,the positive control,oseltamivir (Roche,Switzerland)at the concentration of 25m M (equivalent to 7.109m g/mL),could signi ﬁcantly inhibit the NA activity of H7N9(Fig.2C).Furthermore,the inhibition of H3N2NA activity by rhMBL could also be abolished in the presence of 5mM EDTA or 5mg/mL mannan,suggesting that the NAI was calcium-dependent and mediated by lectin domain.3.4.Steric interference between MBL and NGS in HA and NA head RBD in HA,including the motifs of 190-helix,130-and 220-loop,is one of important functional domains [25].NA activity region is composed of 8functional residues (R118,D151,R152,R224,E276,R292,R371and Y406)and 11framework residues (E119,R156,W178,S179,D198,I222,E227,H274,E277,N294and E425)[18].To elucidate a possible interference between MBL and the NGS around the functional motifs:RBD in HA or the NA activity domain,we compared NGS distribution in them by structure modeling (Fig.S1).We also obtained the trimer form of MBL using the crystal structure (PDB code:1HUP)and measured the radius of MBL CRD which is at least 30Å.Although there are indications that trimers of MBL are not biologically active and at least a tetramer form is needed for activation of complement [26],30Åcan be regard as the minimum value.We subsequently calculated the average distances between the NGS located around RBD or NA activity region (Table 1)and speculated that their distance within the size of human MBL CRD,30Å,might affect the functions of HA or NA.As listed in Table S2and Fig.S1,only one conserved NGS on HA head was detected at position 240in the avian H7N9while different pattern was found in H3and H9(63,122,126,133,144,165,246for H3;95,128,198for H9).The distances of NGS located in H3and H9from its individual RBD all are within 30Åwhile it is of 37.3Åat 240NGS in H7N9.Furthermore,the NGS at residue 165,246[14,27]were indeed associated with the sensitivity of human H3N2virus to MBL and 144for 2009pdm H1N1[28].Notably,additional NGS at Site 133in HA emerged in some H7N9strain (4out of 269)and the effect of MBL on it needed further tests.Similarly,only three NGS at 86,146and 200on NA head were found in H7N9and additional sites including 239,329and 402in H3N2and 239and 402in H9N2.Even the distances of NGS in H7N9NA were less than 30Å,we could not detect any NAI effect of MBL on it.The ﬁndings might attribute to the absence of effective NGS adjacent to NA functional region,such as 239and 402in both H3N2and H9N2virus,or different glycan type attached.4.DiscussionIn ﬂuenza A virus circulating in animal reservoirs is a continual cause for public health concern.In addition to the ever-present threat of seasonal in ﬂuenza,we also face the threats from novel viruses such as H5N1and H7N9,2009pdm H1N1virus in recent years.Since the viruses can escape from the protection of anti-HA or anti-NA antibodies due to antigenic shift,the innate immunity in naïve host is crucial for battling against newly-emerging viruses.MBL,a pattern-recognition molecule in innate immunity,is known as a b inhibitor for seasonal in ﬂuenza A virus.The average concentration of MBL in healthy human plasma (aged 18e 87years)is 1.72±1.51m g/mL [29],with about 30%of individuals present MBL levels below 0.5m g/mL [30].The antiviral activity of MBL against seasonal in ﬂuenza A virus is via its interactions with viral HA or NA by blocking viral entry,fusion or releasing [15,31].MBL could neutralize the virus either in a complement dependent or inde-pendent manner [13,15],However,the effects of MBL may vary depending on speci ﬁc strains.It also plays an important role in modulating in ﬂammation and has been reported to contribute to deleterious in ﬂammatory response to pdmH1N1and a H9N2avian isolate (A/Quail/Hong Kong/G1/97)[32].Here,we found differential binding of rhMBL to human in ﬂu-enza H3N2and avian H7N9,H9N2viruses.Speci ﬁcally,rhMBL exhibited signi ﬁcant HI activity against H3N2and H9N2virus at a relatively low concentration (0.026±0.005,0.083±0.02,respec-tively),while its HI activity on H7N9virus was around 3.67±0.33m g/mL,reaching the upper limit in plasma of healthy population.In contrast,for NAI on H7N9virus,the rhMBL showed little effect even at a high concentration as 10m g/mL.As MBL is supposed to display a steric interference with HA or NA when binding to the speci ﬁc hexose sugars across or adjacent to RBD in HA or activity region in NA,the limited impact of MBL on H7N9might result from its property of few NGS adjacent to functional region on the HA and NA.Of note,a serial of pathotypings including higher virus load,excess chemokine/cytokine responseandFig.2.Inhibition of in ﬂuenza virus Neuraminidase activity by rhMBL.A.Inhibition of H3N2NA activity by rhMBL.Virus was incubated with increasing concentrations of rhMBL (:),or with EDTA at ﬁnal concentration of 5mM (◊),mannan at ﬁnal concentration of 5mg/mL (B ).B.Inhibition of H7N9,H9N2NA activity by rhMBL.H7N9Vac (C )and H9N2RG (-)were incubated with increasing concentrations of rhMBL.NA activity was measured by ELISA,as described in Materials and methods.C.The effects of 10m g/mL rhMBL and 25m M oseltamivir on NA activity of H7N9Vac.Results were expressed as relative NA activity (%)calculated as the OD of the tested wells with virus and rhMBL divided by the OD of the wells only with virus.The dashed line represents 50%of the original NA activity.Data are expressed as mean ±SEM of three independent experiments.*p <0.05.J.Guo et al./Biochemical and Biophysical Research Communications 458(2015)77e 8180functional impairments of B and T cells have been observed in H7N9infection cases,particularly in fatal cases[33,34].The aber-rant inﬂammatory response in H7N9-infected animals could be reversed partially by anti-C5a,indicating a hyperactivated com-plement mediated[35].Therefore,the strong MBL-H7N9virus interaction whereas limited effects on viral HA-receptor binding or NA-mediated releasing,might amplify immune dysfunctions in vivo and confer clinical severity of H7N9infection via activating com-plement pathway and further investigates are needed.Conﬂict of interestNone reported.AcknowledgmentsWe thank National Institute for Biological Standards and Control (UK)and Centers for Disease Control and Prevention(USA)for providing the viruses used in our study.We greatly appreciate Yu Lan for instructions on inﬂuenza bioinformatics.This work was supported by National Mega-projects for Infectious Diseases (2014ZX10004002-001-004).Appendix A.Supplementary dataSupplementary data related to this article can be found at http:// /10.1016/j.bbrc.2015.01.070.Transparency documentThe transparency document associated with this article can be found in the online version at /10.1016/j.bbrc.2015.01.070.References[1]N.Kawasaki,T.Kawasaki,I.Yamashina,Isolation and characterization of amannan-binding protein from human serum,J.Biol.Chem.94(1983) 937e947.[2]h,D.L.Jack,A.W.Dodds,et al.,Mannose-binding lectin binds to a rangeof clinically relevant microorganisms and promotes complement deposition, Infect.Immun.68(2000)688e693.[3]H.Ying,X.Ji,M.L.Hart,et al.,Interaction of mannose-binding lectin with HIVtype1is sufﬁcient for virus opsonization but not neutralization,AIDS Res.Hum.Retrovir.20(2004)327e335.[4]W.E.Ip,K.H.Chan,w,et al.,Mannose-binding lectin in severe acuterespiratory syndrome coronavirus infection,J.Infect.Dis.191(2005) 1697e1704.[5]X.Ji,G.G.Olinger,S.Aris,et al.,Mannose-binding lectin binds to Ebola andMarburg envelope glycoproteins,resulting in blocking of virus interaction with DC-SIGN and complement-mediated virus neutralization,J.Gen.Virol.86 (2005)2535e2542.[6]P.Fischer,S.Ellermann-eriksen,S.Thiel,et al.,Mannan-binding protein andbovine conglutinin mediate enhancement of herpes simplex virus type2 infection in mice,Scand.J.Immunol.39(1994)439e445.[7]W.Eddie Ip,K.Takahashi,R.Alan Ezekowitz,et al.,Mannose-binding lectinand innate immunity,Immunol.Rev.230(2009)9e21.[8]R.A.Medina, A.García-Sastre,Inﬂuenza A viruses:new research de-velopments,Nat.Rev.Microbiol.9(2011)590e603.[9]K.Y.Yuen,P.K.Chan,M.Peiris,et al.,Clinical features and rapid viral diagnosisof human disease associated with avian inﬂuenza A H5N1virus,Lancet351 (1998)467e471.[10]M.Peiris,K.Yuen,C.Leung,et al.,Human infection with inﬂuenza H9N2,Lancet354(1999)916e917.[11]R.A.Fouchier,P.M.Schneeberger,F.W.Rozendaal,et al.,Avian inﬂuenza Avirus(H7N7)associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome,Proc.Natl.Acad.Sci.101(2004)1356e1361.[12]H.Chen,H.Yuan,R.Gao,et al.,Clinical and epidemiological characteristics of afatal case of avian inﬂuenza A H10N8virus infection:a descriptive study, Lancet383(2014)714e721.[13] E.M.Anders, C.A.Hartley,P.C.Reading,et al.,Complement-dependentneutralization of inﬂuenza virus by a serum mannose-binding lectin,J.Gen.Virol.75(1994)615e622.[14]K.L.Hartshorn,K.Sastry,M.White,et al.,Human mannose-binding proteinfunctions as an opsonin for inﬂuenza A viruses,J.Clin.Invest.91(1993)1414.[15]T.Rase,Y.Suzuki,T.Kawai,et al.,Human mannan-binding lectin inhibits theinfection of inﬂuenza A virus without complement,Immunology97(1999) 385e392.[16]R.Gao,B.Cao,Y.Hu,et al.,Human infection with a novel avian-origin inﬂu-enza A(H7N9)virus,N.Engl.J.Med.368(2013)1888e1897.[17]J.Zhou,D.Wang,R.Gao,et al.,Biological features of novel avian inﬂuenza A(H7N9)virus,Nature499(2013)500e503.[18]W.Zhu,Y.Zhu,K.Qin,et al.,Mutations in polymerase genes enhanced thevirulence of2009pandemic H1N1inﬂuenza virus in mice,PLoS ONE7(2012) e33383.[19]G.K.Hirst,Adsorption of inﬂuenza hemagglutinins and virus by red bloodcells,J.Exp.Med.76(1942)195e209.[20]World Health Organization.Manual for the laboratory diagnosis and viro-logical surveillance of inﬂuenza,http://www.who.int/inﬂuenza/gisrs_ laboratory/manual_diagnosis_surveillance_inﬂuenza/en/;[accessed on19.12.2014].[21] mbr e,H.Terzidis,A.Greffard,et al.,Measurement of anti-inﬂuenzaneuraminidase antibody using a peroxidase-linked lectin and microtitre plates coated with natural substrates,J.Immunol.Methods.135(1990) 49e57.[22]M.Biasini,S.Bienert,A.Waterhouse,et al.,SWISS-MODEL:modelling proteintertiary and quaternary structure using evolutionary information,Nucleic.Acids.Res.42(2014)W252e W258.[23] E.Krissinel,K.Henrick,Inference of macromolecular assemblies from crys-talline state,J.Mol.Biol.372(2007)774e797.[24] D.Kobasa,M.E.Rodgers,K.Wells,et al.,Neuraminidase hemadsorption ac-tivity,conserved in avian inﬂuenza A viruses,does not inﬂuence viral repli-cation in ducks,J.Virol.71(1997)6706e6713.[25]I.A.Wilson,J.J.Skehel,D.C.Wiley,Structure of the haemagglutinin membraneglycoprotein of inﬂuenza virus at3A resolution,Nature289(1981)366e373.[26]O.Ferraris,B.Lina,Mutations of neuraminidase implicated in neuraminidaseinhibitors resistance,J Clin Virol41(2008)13e19.[27]P.C.Reading,D.L.Pickett,M.D.Tate,P.G.Whitney,E.R.Job,A.G.Brooks,Loss ofa single N-linked glycan from the hemagglutinin of inﬂuenza virus is associ-ated with resistance to collectins and increased virulence in mice,Respir.Res.10(2009)44.[28]M.D.Tate,A.G.Brooks,P.C.Reading,Speciﬁc sites of N-linked glycosylation onthe hemagglutinin of H1N1subtype inﬂuenza A virus determine sensitivity to inhibitors of the innate immune system and virulence in mice,J.Immunol.187(2011)1884e1894.[29]T.Yoshizaki,K.Ohtani,W.Motomura,et al.,Comparison of human bloodconcentrations of collectin kidney1and mannan-binding lectin,J.Biol.Chem.151(2012)57e64.[30]K.A.Petersen,F.Matthiesen,T.Agger,et al.,Phase I safety,tolerability,andpharmacokinetic study of recombinant human mannan-binding lectin,J.Clin.Immunol.26(2006)465e475.[31] E.Leikina,H.Delanoe-Ayari,K.Melikov,et al.,Carbohydrate-binding mole-cules inhibit viral fusion and entry by crosslinking membrane glycoproteins, Nat.Immunol.6(2005)995e1001.[32]M.T.Ling,W.Tu,Y.Han,et al.,Mannose-binding lectin contributes to dele-terious inﬂammatory response in pandemic H1N1and avian H9N2infection, J.Infect.Dis.205(2012)44e53.[33]H.-N.Gao,H.-Z.Lu,B.Cao,et al.,Clinicalﬁndings in111cases of inﬂuenza A(H7N9)virus infection,N.Engl.J.Med.368(2013)2277e2285.[34]W.Wu,Y.Shi,H.Gao,et al.,Immune derangement occurs in patients withH7N9avian inﬂuenza,Crit.Care.18(2014).R43.[35]S.Sun,G.Zhao,C.Liu,et al.,Treatment with anti-C5a antibody improves theoutcome of H7N9virus infection in African green monkeys,Clin.Infect.Dis.60 (2015)586e595.J.Guo et al./Biochemical and Biophysical Research Communications458(2015)77e8181。

杏仁核-海马环路参与抑郁症的发病机制

· 93 ·中国疼痛医学杂志Chinese Journal of Pain Medicine 2021, 27 (2)[49] Teodor, Goroszeniuk. The effect of peripheral neuro-modulation on pain from the sacroiliac joint: A retro-spective cohort study[J]. Neuromodulation: Journal of the International Neuromodulation Society, 2019, 22(5):661-666.[50] Kim YH, Moon DE. Sacral nerve stimulation for thetreatment of sacroiliac joint dysfunction: A case re-port[J]. Neuromodulation, 2010, 13(4):306-310. [51] Dengler J, Kools D, Pﬂugmacher R, et al. Randomizedtrial of sacroiliac joint arthrodesis compared with con-servative management for chronic low back pain at-tributed to the sacroiliac joint[J]. J Bone Joint Surg Am, 2019, 101(5): 400-411.[52] Sachs D, Capobianco R. One year successful outcomesfor novel sacroiliac joint arthrodesis system[J]. Ann Surg Innovat Res, 2012, 6(1):13.[53] Smith AG, Capobianco R, Cher D, et al. Open versusminimally invasive sacroiliac joint fusion: A multi-center comparison of perioperative measures and clinical outcomes[J]. Ann Surg Innovat Res, 2013, 7(1):14.[54] Capobianco R, Cher D. Safety and effectiveness ofminimally invasive sacroiliac joint fusion in women with persistent post-partum posterior pelvic girdle pain: 12-month outcomes from a prospective, multi-center trial[J]. SpringerPlus, 2015, 4:570.•国际译文•杏仁核-海马环路参与抑郁症的发病机制慢性应激常可引发抑郁症。

国家人类基因组北方研究中心

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Functional Proteomics – To study protein-protein interaction, 3-D structures, cellular localization and PTMs in order to understand the physiological function of the whole set of proteome.
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JBC-细胞核蛋白提取参考文献

Nerve Growth Factor Stimulates Proliferation and Survival of Human Breast Cancer Cells through Two Distinct Signaling Pathways*Received for publication,November 20,2000,and in revised form,February 14,2001Published,JBC Papers in Press,February 28,2001,DOI 10.1074/jbc.M010499200Simon Descamps‡§,Robert-Alain Toillon‡,Eric Adriaenssens§¶,Vale ´rie Pawlowski§ʈ,Simon M.Cool**,Victor Nurcombe**,Xuefen Le Bourhis‡,Be ´noni Boilly‡,Jean-Philippe Peyrat ʈ,and Hubert Hondermarck‡‡‡From the ‡Equipe Facteurs de Croissance,UPRES EA-1033Biologie du De ´veloppement,Universite ´des Sciences et Technologies de Lille,59655Villeneuve d’ASCQ France,the ¶Immunopathologie Cellulaire des Maladies Infectieuses,CNRS,UMR 8527,Institut de Biologie de Lille,59000France,the **Department of Anatomical Sciences,University of Queensland,St.Lucia,Queensland 4072,Australia,and the ʈLaboratoire d’Oncologie Mole ´culaire Humaine,Centre Oscar Lambret,59020Lille,FranceWe show here that the neurotrophin nerve growth factor (NGF),which has been shown to be a mitogen for breast cancer cells,also stimulates cell survival through a distinct signaling pathway.Breast cancer cell lines (MCF-7,T47-D,BT-20,and MDA-MB-231)were found to express both types of NGF receptors:p140trkA and p75NTR .The two other tyrosine kinase receptors for neu-rotrophins,TrkB and TrkC,were not expressed.The mitogenic effect of NGF on breast cancer cells required the tyrosine kinase activity of p140trkA as well as the mitogen-activated protein kinase (MAPK)cascade,but was independent of p75NTR .In contrast,the anti-apo-ptotic effect of NGF (studied using the ceramide ana-logue C2)required p75NTR as well as the activation of the transcription factor NF-kB,but neither p140trkA nor MAPK was necessary.Other neurotrophins (BDNF,NT-3,NT-4/5)also induced cell survival,although not proliferation,emphasizing the importance of p75NTR in NGF-mediated survival.Both the pharmacological NF-␬B inhibitor SN50,and cell transfection with IkBm,resulted in a diminution of NGF anti-apoptotic effect.These data show that two distinct signaling pathways are required for NGF activity and confirm the roles played by p75NTR and NF-␬B in the activation of the survival pathway in breast cancer cells.Nerve growth factor (NGF)1is the archetypal member of theneurotrophin superfamily,which also includes brain-derived neurotrophic factor (BDNF),neurotrophin-3(NT-3),NT-4/5,and NT-6(1).NGF interacts with two classes of membrane receptor:the TrkA proto-oncogene product p140trkA ,which pos-sesses intrinsic tyrosine kinase activity,and a secondary re-ceptor,p75NTR ,that belongs to the tumor necrosis factor (TNF)receptor family (2).The stimulation of cell survival and cell differentiation by NGF and other neurotrophins have been described primarily in neuronal cell systems (3).Although the neurotrophic effect through p140trkA is known to involve the MAPK cascade,the role of p75NTR is still controversial;there is evidence that it can both positively and negatively regulate neuronal cell death and differentiation,depending on the cell type examined (4).In some cases,p75NTR is an inducer of apoptosis,even without NGF stimulation (5),whereas in other cases the activation of p75NTR by NGF results in a protection from cell death (6).In addition to its neurotrophic function,other activities of NGF have been described.For example,NGF can modulate gene expression in monocytes (7),it is chemotac-tic for melanocytes (8),and its inhibition on p75NTR can block the migration of Schwann cells (9).NGF also stimulates the proliferation of chromaffin cells (10),lymphocytes (11),and keratinocytes (12).We have previously shown that NGF is mitogenic for cancerous but not normal human breast cells (13),and these data,as well as others showing a role for NGF in the stimulation of prostatic cancer cells (14–17),implicate NGF in non-neuronal carcinogenesis.Both cellular proliferation as well as tumor cell survival are crucial for malignant progression.The effect of NGF on the survival of cancer cells through the p75NTR receptor has been shown for neuroblastoma (18)and schwannoma (6).In prostate cancer,p75NTR has been shown to be a mediator of NGF’s effects during critical phases of developmental cell death and carcinogenic progression (19).To date only the mitogenic effect of NGF for breast cancer cells has been described (13),with its roles in the control of breast cancer cell survival unknown.In this study,we have shown that,in addition to its mito-genic effect,NGF is also an anti-apoptotic factor for breast cancer cells.These cells express mRNA for both p140trkA and p75NTR receptors.Our results indicate that the mitogenic effect of NGF requires p140trkA and the MAPK cascade,but not the p75NTR receptor,whereas the promotion of cell survival strictly requires p75NTR as well as NF-␬B,but not p140trkA and MAPK.Thus the mitogenic and anti-apoptotic effects of NGF on breast*This work was supported in part by a grant from the Ligue Na-tionale Contre le Cancer (Comite ´du Nord)and by the French Ministry of Research and Education.The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked “advertisement ”in accordance with 18U.S.C.Section 1734solely to indicate this fact.§Recipients of an Association pour la Recherche sur la Cancer fellowship.‡‡To whom correspondence should be addressed:EA-1033,batiment SN3,Universite ´des Sciences et Technologies de Lille,59655Villeneuve d’Ascq cedex,France.Tel.:33-3-20-43-40-97;Fax:33-3-20-43-40-38;E-mail:hubert.hondermarck@univ-lille1.fr.1The abbreviations used are:NGF,nerve growth factor;PAGE,poly-acrylamide gel electrophoresis;NF-␬B,nuclear factor-␬B;BDNF,brain-derived neurotrophic factor;NT,neurotrophin;PARP,polyADP-ribose polymerase;TNF,tumor necrosis factor;TBP,TATA box binding pro-tein;RT-PCR,reverse transcriptase-polymerase chain reaction;FCS,fetal calf serum;DTT,dithiothreitol;PBS,phosphate-buffered saline;ERK,extracellular signal-regulated kinase;GFP,green fluorescence protein;I ␬Bm,dominant-negative I ␬B ␣mutant;bp,base pair(s);PD98059,Park Davis 98059.T HE J OURNAL OF B IOLOGICAL C HEMISTRYVol.276,No.21,Issue of May 25,pp.17864–17870,2001©2001by The American Society for Biochemistry and Molecular Biology,Inc.Printed in U.S.A.This paper is available on line at 17864cancer cells are mediated through two different signaling pathways.EXPERIMENTAL PROCEDURESMaterials—Cell culture reagents were purchased from BioWhittaker (France)except insulin,which was obtained from Organon(France). Recombinant human nerve growth factor,brain derived growth factor (BDNF),and neurotrophins3(NT-3)and4(NT-4)were from R&D Systems(UK).K-252a(inhibitor of trk-tyrosine kinase activity)and PD98059(inhibitor of MAPK cascade)were from Calbiochem(France). The mouse monoclonal anti-NGF receptor(p75NTR)antibody was from Euromedex(France)and was previously described for its ability to block the interaction between p75NTR and NGF(20).The anti-lamin B(C-20), goat polyclonal IgG,and the polyclonal anti-p140trkA(trk763)were from Santa Cruz Biotechnology.C2ceramide analogue(N-acetyl-D-sphingo-sine),Hoechst33258,and electrophoresis reagents were from Sigma Chemical Co.(France).The SN50NF-␬B inhibitor peptide,the rabbit polyclonal anti-NF-␬B p65antibody,was obtained from TEBU(France). Anti-PARP antibody was from Oncogene Research Products(UK). Primers and probes for TrkA and p75NTR,probe for TATA box binding protein(TBP)were from Eurogentec(Belgium).RT-PCR reagents were from Applied Biosystems(France).Lipofectin reagent and Opti-MEM were provided by Life Technologies,Inc.(France).The green fluores-cence protein plasmid(EGFPC1)was purchased from CLONTECH,and the dominant-negative I␬B␣mutant(I␬Bm)expression vectors(in PCDNA3)containing a Ser to Ala substitution at residues32and36 were obtained from Dr.Jean Feuillard(UPRES EA1625,Bobigny, France).p65(rel-A)and c-rel cDNA were cloned at Eco RI site in PSVK3 expression plasmid.All vectors were obtained from Dr.Pascale Cre´pieux(McGill University,Montreal).The SY5Y subclone of SK-N-SH neuroblastoma cell line was a kind gift of Dr.Luc Bue´e(INSERM, U422,Lille,France).NT-2(Ntera/D1)human neural precursor cells (Stratagene)are derived from a clone of the NT-2teratocarcinoma.Cell Culture—Breast cancer cell lines(MCF-7,T47-D,BT-20,and MDA-MB-231)were obtained from the American Type Culture Collec-tion and routinely grown as monolayer cultures.Cells were maintained in minimal essential medium(Earle’s salts)supplemented with20m M Hepes,2g/liter sodium bicarbonate,2m M L-glutamine,10%fetal calf serum(FCS),100units/ml penicillin-streptomycin,50␮g/ml gentami-cin,1%of non-essential amino acids,and5␮g/ml insulin.Detection of Neurotrophin Receptors mRNA Expression—The reverse transcription reaction mixture contained2g of purified total RNA (extracted from breast cancer cell lines,NT-2cells,or SY5Y cells),1ϫreverse transcription reaction buffer,10m M DTT,400m M dNTP each, 2.5M oligo(dT)18primer,40units of RNasin,and200units of Moloney murine leukemia virus reverse transcriptase were added to25␮l of total reaction volume.All the reaction mixtures were incubated at37°C for1h and then inactivated at95°C for5min.Polymerase chain reaction was performed on cDNAs after RT or corresponding total RNA samples without the RT step for negative controls.The primers used for trkA and p75RT-PCR detection in breast cancer cell lines were as follows:trkA sense primer,5Ј(291)-CATCGTGAAGAGTGTCTCCG-3Ј(311)and antisense primer,5Ј(392)-GAGAGAGACTCCAGAGCGTT-GAA-3Ј(370)or p75sense primer,5Ј(442)-CCTACGGCTACTACCAG-GATGAG-3Ј(462)and antisense primer,5Ј(588)-TGGCCTCGTCG-GAATACG-3Ј(571).The primers used for RT-PCR comparative detection of trks in MCF-7cells were as follows:trkA sense primer,5Ј(118)-AGGCGGTCTGGTGACTTCGTTG-3Ј(139)and antisense primer, 5Ј(1162)-GGCAGCCAGCAGGGTGTAGTTC-3Ј(1141)or trkB sense primer,5Ј(134)-CGAGGTTGGAACCTAACAGCATTG-3Ј(157)and an-tisense primer,5Ј(1182)-GTCAGTTGGCGTGGTCCAGTCTTC-3Ј(1159)or trkC sense primer,5Ј(219)-CACGGACATCTCAAGGAAGA-GCA-3Ј(241)and antisense primer,5Ј(1078)-CTGAGAACTTCACCC-TCCTGGTAG-3Ј(1056).Each pair of primers was used in RT-PCR reaction to amplify trks or p75.To PCR tubes were added5␮l of PCRbuffer(200m M Tris-HCl,pH8.4,500m M KCl),10␮l of15m M MgCl2,1␮l of10m M dNTP mix,1␮l of cDNA or total mRNA(for negative control),1␮l of50m M respective primers,1␮l of2.5units/␮l Taq DNA polymerase,and water to a total volume of50␮l.The PCR conditions were as follows:after95°C for3min for denaturing cDNA,30cycles were run at94°C for1min,57°C for2min,and72°C for3min.The PCR tubes were incubated for a further10min at72°C for the exten-sion of cDNA fragments after the final cycle,and the PCR products were electrophoresed in an agarose gel.Cell Growth Assay—Experiments were performed as previously de-scribed(13).35-mm diameter dishes were inoculated with2ϫ104 cells/dish in2ml of medium containing10%FCS.After24h,cells were washed twice with serum-free medium.Next day,the medium wasreplaced with2ml of serum free medium containing100ng/ml NGF orvarious concentrations of other neurotrophins(BDNF,NT-3,NT-4/5).To study the effect of pharmacological inhibitors or blocking antibodies,various concentrations were added simultaneously with NGF(100ng/ml).After2days of NGF exposure,cells were harvested by trypsiniza-tion and counted using an hemocytometer.Determination of the Percentage of Apoptotic Cell Nuclei—Apoptosisof breast cancer cells was induced by the ceramide analogue C2,whichhas been described as a pro-apoptotic agent for human breast cancercells(21,22).Apoptosis was obtained by treatment with2␮M C2for 24h.To evaluate the anti-apoptotic activity of NGF,various concen-trations of this factor were tested;we found that the maximal effect wasobtained for100ng/ml.Consequently,this concentration was used in allexperiments with pharmacological inhibitors or blocking antibody.Fordetermination of apoptotic cell percentage,cells were fixed with coldmethanol(Ϫ20°C)for10min and washed twice with phosphate-buff-ered saline(PBS)before staining with1␮g/ml Hoechst33258for10min at room temperature in the dark.Cells were then washed with PBS andmounted with coverslips using Glycergel(Dako).The apoptotic cellsexhibiting condensed and fragmented nuclei were counted under anOlympus-BH2fluorescence microscope in randomly selected fields.Aminimum of500–1000cells was examined for each condition,andresults were expressed as a ratio of the total number of cells counted.Statistical Analysis and Software—The statistical analysis of thedata gathered from cell and apoptotic nuclei counting was performedusing SPSS version9.0.1(SPSS inc.,Chicago,IL).Analyses of variancewere followed by the Tukey’s test to determine the significance.NGF Receptors and PARP Immunoblotting—Subconfluent cell cul-tures were harvested by scraping in serum-free medium.After centrif-ugation(1000ϫg,5min),the pellet was treated with lysis buffer(0.3%SDS,200m M dithiothreitol)and boiled5min.In the case of PARP,thepellet was lysed with urea-rich buffer(62.5m M Tris-HCl,pH6.8,6Murea,10%glycerol,2%SDS),sonicated and incubated at65°C for15min.The lysates were subjected to SDS-PAGE,transferred onto anitrocellulose membrane(Immobilon-P,Millipore)by electroblotting(100V,75min),and probed with anti-trkA,anti-p75NTR or anti-PARPantibodies at4°C overnight.The membranes were then incubated atroom temperature for3h with biotin-conjugated anti-rabbit(TrkA)oranti-mouse(p75NTR and PARP)immunoglobulin G.After1h of incu-bation with extravidin,the reaction was revealed using the chemilumi-nescence kit ECL(Amersham Pharmacia Biotech)with Kodak X-OmatAR film.Detection of p140trkA and MAPK Activation—Proteins were extractedin lysis buffer(150m M NaCl,50m M Tris,pH7.5,0.1%SDS,1%NonidetP-40,100␮M sodium orthovanadate)prior to immunoprecipitation. Preclearing was done with protein A-agarose(10␮l/250␮l,60min, 4°C).After centrifugation(10,000ϫg,2min),the supernatant wasincubated with monoclonal anti-MAPK(anti-ERK2)antibody(10␮l/250␮l,60min,4°C).Protein A-agarose(10␮l)was added for60min(4°C) and then pelleted by centrifugation(10,000ϫg,2min).The pellet wasthen rinsed three times with lysis buffer and boiled for5min inLaemmli buffer.After SDS-PAGE and electroblotting,nitrocellulosemembranes were blocked with3%bovine serum albumin.Membraneswere then incubated with PY20anti-phosphotyrosine antibody over-night at4°C,rinsed,and incubated with a horseradish peroxidase-conjugated anti-mouse IgG for3h at room temperature.Membraneswere rinsed overnight at4°C before visualization with ECL.Cell Fractionation and NF-␬B Detection—Cell nuclear extracts were prepared as described by Herrmann et al.(23).Cells were trypsinized and then pelleted in minimal essential medium containing10%FCS. After washing with ice-cold PBS,cells were repelleted and resuspended in400␮l of ice-cold hypotonic buffer(10m M Hepes,pH7.8,10m M KCl,2m M MgCl2,0.1m M EDTA,10␮g/ml aprotinin,0.5␮g/ml leupeptin,3 m M phenylmethylsulfonyl fluoride,and3m M DTT).After10min on ice, 25␮l of10%Nonidet P-40was added and crude nuclei were collected by centrifugation for5min.The nuclear pellet was resuspended in high salt buffer(50m M Hepes,pH7.4,50m M KCl,300m M NaCl,0.1m M EDTA,10%(v/v)glycerol,3m M DTT,and3m M phenylmethylsulfonyl fluoride).After30min on ice with frequent agitation,the insoluble nuclear material was pelleted in a microcentrifuge for10min.Crude nuclear protein was collected from the supernatant and snap-frozen in a dry ice/ethanol bath.After thawing and boiling for5min in Laemmli buffer,the nuclear extracts were subjected to SDS-PAGE and probed with an anti-NF-␬B p65antibody.A control was established with anti-lamin B antibody.Transfection of I␬␤,c-rel,and rel-A—Cotransfection experiments were carried out using Lipofectin reagent,as described by the manu-Intracellular Signaling Pathways of NGF in Breast Cancer Cells17865facturer.Briefly,MCF-7cells were incubated for 5h in 1ml of Opti-MEM transfection medium containing 8␮l of Lipofectin reagent,0.8␮g of green fluorescence protein (GFP)-carrying vector and 0.2␮g of empty vector PCDNA3or 0.2␮g of I ␬Bm.In the case of c-rel or rel-A,cells were cotransfected with 0.8␮g of GFP-carrying GFP and 0.6␮g of PSVK 3(empty plasmid),c-rel,or rel-A.Cells were then grown for 24h with 10%FCS minimal essential medium and rinsed for 2h in serum-free me-dium before incubation in serum-free medium in the presence or ab-sence of 100ng/ml NGF and/or 2␮M C2for another 24h.Cells were then fixed with paraformaldehyde 4%(4°C)for 30min,and the per-centage of apoptotic cell nuclei in GFP-stained cells was determined as described above.RESULTSNGF Mitogenic and Anti-apoptotic Activity for Breast Cancer Cells—The effects of 100ng/ml NGF on cell proliferation and C2-induced apoptosis were evaluated by cell counting and Hoechst staining,respectively.The results show that NGF induces an increase in cell number for all breast cancer cell lines tested (Fig.1A ).We have previously demonstrated that NGF has a direct mitogenic effect on breast cancer cells by recruiting cells in G 0phase and by shortening the G 1length (Descamps et al.,1998).In addition,NGF rescued breast cancer cells undergoing C2-induced apoptosis;the maximum survival was observed at 200ng/ml (Fig.1B ).The morphology of cells undergoing this NGF-induced anti-apoptotic rescue was quite distinct (Fig.2A ).The induction of apoptosis by C2was found to involve cleavage of poly(A)DP-ribose polymerase (PARP);this cleavage was reversed by NGF (Fig.2B ).TrkA and p75NTR Expression—RT-PCR was used to show the expression of mRNA for both high and low affinity NGF recep-tors in MCF-7,T47-D,BT-20,and MDA-MB-231cells (Fig.3A );the 102-bp band for the TrkA transcript and a 147-bp band for the p75NTR transcript were readily detectable on 1%agarose gels.Moreover,Western blotting demonstrated that both p140trkA and p75NTR were present in all the breast cancer cell lines (Fig.3B ).Real-time quantitative RT-PCR indicated that there was no significant change in the levels of TrkA and p75NTR mRNAs in the presence of FCS,NGF,or C2(data not shown)and that the levels of mRNA for TrkA and p75NTR in breast cancer cells was between 5and 10times lower than the level observed in SY5Y neuroblastoma cells (data not shown).This indicates that NGF receptor expression in breast cancer cells is relatively limited.It should be noticed that,although mRNA levels of NGF receptors differ between breast cancer cells and SY5Y,the protein levels apparently do not.However,F IG .1.Effect of NGF on the growth and survival of breast cancer cells.A ,breast cancer cells were serum-deprived in minimum essential medium,and after 24h the NGF (100ng/ml)was added.After 48h,cells were harvested and counted.B ,cells were serum-deprived in minimum essential medium and treated with 2␮M C2with or without 100ng/ml NGF.After 24h,cells were fixed and the proportion of apoptotic nuclei were determined after Hoechst staining under an Olympus-BH2fluorescence microscope.For measurement of both cell number and apoptosis,results are expressed as the means ϮS.D.of five separate experiments.Significance was determined using the Tukey’s test (*,p Ͻ0.01).F IG .2.Anti-apoptotic effect of NGF.A ,Hoechst staining of apop-totic cell nuclei in control,C2and C2ϩNGF-treated MCF-7cells.Cells were serum-deprived in minimum essential medium and treated with C2.NGF was added at 100ng/ml.After 24h,cells were fixed and apoptotic nuclei were observed after Hoechst staining.B ,immunoblot detection of PARP cleavage.C2-induced PARP cleavage was reversed by p75NTR activation mediated by NGF.MCF-7cells were serum-de-prived in minimum essential medium for 24h and were then treated with 100ng/ml NGF in the presence or absence of 2␮M C2,10n M K-252a,10␮M PD98059,or 10␮g/ml anti-p75NTR -blocking antibody (Euromedex)for another 24-h period.Proteins were detected after SDS-PAGE of cell preparations from MCF-7breast cancer cells,electro-blotting onto nitrocellulose,and immunodetection with anti-PARP antibodies.Intracellular Signaling Pathways of NGF in Breast Cancer Cells17866it has been shown before that the level of a given cellular protein cannot be simply deduced from mRNA transcript level (24).One could hypothesize that the stability of mRNA and/or protein for NGF receptors,differs between breast cancer cells and neuroblastoma cells,leading to the observed disproportion-ality between mRNA and protein levels.Involvement of p140trkA and p75NTR in Mitogenic and Sur-vival Activities of NGF—We used a combination of specific antibodies and pharmacological inhibitors to study the puta-tive functions of p140trkA and p75NTR in the stimulation of proliferation and cell survival induced by NGF.The Trk tyro-sine kinase inhibitor K-252a,and the MEK inhibitor PD98059,both strongly inhibited the growth-stimulatory effect of NGF on MCF-7cells,but had no effect on its anti-apoptotic effects (Fig.4).Conversely,neither the anti-p75NTR blocking antibody nor the NF-␬B inhibitor SN50affected NGF-stimulated prolif-eration,although both strongly reduced the anti-apoptotic ef-fects (Fig.4).The tyrosine kinase activity of p140trkA was inhibited by K-252a but not by the anti-p75NTR or PD98059(Fig.5).On the other hand,the activity of the MAPKs was inhibited by K-252a and PD98059but not by the anti-p75NTR (Fig.5).It should be noted that the SN50peptidic inhibitor of NF-␬B,similarly to the anti-p75NTR ,inhibited the anti-apo-ptotic effect of NGF but neither its proliferative effect nor its activation of p140trkA and MAPKs.The effect of other neuro-trophins on MCF-7cell growth and survival was also evaluated (Fig.6A ).In contrast to NGF,no proliferative effect was pro-vided by BDNF,NT-3,or NT-4/5.However,all neurotrophins tested exhibited a rescue effect on C2-treated cells that was not altered in the presence of the trk inhibitor K-252a (Fig.6B ).These data suggest that trk receptors are not involved in NGF survival activity.Moreover,the participation of trkB and trkCin these events can be ruled out,because they are not expressed in these breast cancer cells (Fig.6C ).NF-␬B Involvement in the Anti-apoptotic Effect of NGF—The inhibitory effect of SN50on the NGF anti-apoptotic activity indicated the potential involvement of NF-␬B in the signaling leading to the protective activity of this growth factor.To fur-ther investigate this phenomenon,we studied the effect of NGF on the nuclear translocation of NF-␬B,as well as the conse-quence of transfection by IkBm (an inhibitor of NF-␬B)or by c-rel and rel-A (constitutively active subunits of NF-␬B)on the NGF-mediated anti-apoptotic activity in MCF-7cells.Western blotting revealed no change in the nuclear levels of NF-␬B (p65)during apoptosis induced by C2(Fig.7).In contrast,the addi-tion of NGF on C2-treated cells induced a translocation of NF-␬B from cytoplasm to puterized quantifica-tion revealed a doubling p65band intensity normalized to the total intensity of the lane (data not shown).Moreover,this NF-␬B nuclear translocation was inhibited by the presence of p75NTR -blocking antibody or SN50,but was not affected by K-252a and PD98059.Interestingly,in the absence of C2-in-duced apoptosis NGF was not able to induce the nuclear trans-location of NF-␬B,confirming previous observations that p75NTR -mediated NF-␬B activation requires cell stress (25).Transfection of MCF-7cells with IkBm,an inhibitor of NF-␬B,reversed the anti-apoptotic effect of NGF (Fig.8A ).As a control,we transfected MCF-7cells with an empty vector;no effect was observed.In addition,transfection with activators of the NF-␬B pathway,c-rel or rel-A (Fig.8B ),resulted in an inhibition of C2-induced apoptosis of MCF-7cells,even in absence ofNGF,F IG .3.TrkA and p75NTR expression in breast cancer cells.A ,agarose gel electrophoresis of RT-PCR products evidenced a 102-bp band and a 147-bp band,which are characteristic of TrkA and p75NTR ,respectively.Both NGF receptors were found in all cell types tested.B ,p140trkA and p75NTR were immunodetected after SDS-PAGE of breast cancer cell lines.The neuroblastoma cells SY5Y were used as positive control for the expression of NGFreceptors.F IG .4.Pharmacological modulation of the proliferative and anti-apoptotic effect of NGF.MCF-7cells were starved in minimum essential medium,and after 24h,100ng/ml NGF was added with or without inhibitors or antibody.A ,after 48h,cells were harvested and counted.B ,after 24h,cells were fixed and the proportion of apoptotic nuclei determined after Hoechst staining.The following concentrations were used:2␮M C2,10n M K-252a,10␮g/ml anti-p75NTR -blocking antibody (Euromedex),10␮M PD98059,18␮M SN50.For A and B ,results are expressed as the means ϩS.D.of five separate experiments.Significance was determined using the Tukey’s test (*,p Ͻ0.01).Intracellular Signaling Pathways of NGF in Breast Cancer Cells 17867confirming the involvement of NF-␬B family members in hu-man breast cancer cell survival.DISCUSSIONThis study shows that,in addition to its mitogenic activity,NGF is anti-apoptotic for breast cancer cells,and that these two biological effects are differentially mediated by the p140trkA and p75NTR receptors,respectively.The growth of breast cancer results from a balance between cell proliferation and apoptosis,both of which can be modulated by various regulatory peptides.For example,epidermal growth factor,fibroblast growth factors,and insulin-like growth factor-1can all stimulate the proliferation and survival of breast cancer cells (26).On the other hand,agents such as transforming growth factor-␤or tumor necrosis factor-␣can inhibit growth and induce apoptosis in these cells (27).Recently we have shown that NGF,which was primarily described for its neuro-trophic properties,is a strong mitogen for cancerous but not for normal human breast epithelial cells,suggesting a crucial func-tion for this factor in the initiation and progression of human breast tumors (13).In the present study,we have shown that the breast cancer cells express transcripts for both TrkA and p75NTR receptors.In contrast,no expression of TrkB and TrkC was found in any of the breast cancer cells tested,in accordance with the fact that BDNF,NT-3,or NT-4/5have no mitogenic effect for these cells.The presence of NGF receptors has been detected previously in breast cancer cells (28),and low levels of NGF receptor expression have recently been reported in other breast cancer cell lines (29),leading to the hypothesis of a recruitment and cooperation between p140trkA and p185Her-2for the induction of mitogenesis by NGF.Our results indicate a stimulation of p140trkA tyrosine kinase activity and of the MAPK cascade by NGF,and the use of the pharmacological inhibitors K-252a and PD98059demonstrate the requirementfor these signals in NGF-induced MCF-7cell proliferation.The induction of MAPK activity required p140trkA activation,but p75NTR did not appear to be involved,because p75NTR -blocking antibodies did not have any effect on NGF-inducedMAPKF IG .5.p140trkA and MAPK activation.MCF-7cells were treated with 100ng/ml NGF in the presence or absence of 10n M K-252a,10␮g/ml anti-p75NTR -blocking antibody,or 10␮M PD98059.p140trkA (A )and MAPK activation (B )were determined after immunoprecipitation using polyclonal anti-TrkA and monoclonal anti-ERK2antibodies,re-spectively.After SDS-PAGE and electroblotting,nitrocellulose mem-branes were counterprobed with the PY20anti-phosphotyrosine anti-body.For detection of TrkA (A )and MAPK (B )activation,the lower panel shows reprobing of the blots with the immunoprecipitatingantibody.F IG .6.Effect of different neurotrophins on MCF-7cells growth and survival.MCF-7cells were serum-deprived in minimum essential medium,and after 24h the neurotrophins (100ng/ml NGF,50ng/ml BDNF,50ng/ml NT-3,100ng/ml NT-4/5)were added.A ,after 48h,cells were harvested and counted.In contrast with NGF,neither BDNF,NT-3,nor NT-4/5displayed significant bioactivity (for concen-trations up to 400ng/ml).B ,MCF-7cells were serum-deprived in minimum essential medium and treated with 2␮M C2,with or without neurotrophins (100ng/ml NGF,50ng/ml BDNF,50ng/ml NT-3,100ng/ml NT-4/5).After 24h,cells were fixed and apoptotic nuclei percent-age was determined after Hoechst staining under an Olympus-BH2fluorescence microscope.For measurement of both cell number and apoptosis,results are expressed as the means ϮS.D.of five separate experiments.Significance was determined using the Tukey’s test (*,p Ͻ0.01).C ,TrkB and TrkC mRNA expression in MCF-7cells.Agarose gel electrophoresis of RT-PCR products reveals TrkA expression,but no TrkB or TrkC expression in MCF-7breast cancer cells.Human NT2cells were used as positive control for the expression of TrkB and ne 1,NT2-negative control without RT step;lane 2,NT2-positive control;lane 3,MCF-7cells-negative control without RT step;lane 4,MCF-7cells.Intracellular Signaling Pathways of NGF in Breast Cancer Cells17868。

以癫痫起病及DNAJC5基因突变的成人型神经元蜡样脂褐质沉积症家系

以癫痫起病及DNAJC5基因突变的成人型神经元蜡样脂褐质沉积症家系以癫痫起病及DNAJC5基因突变的成人型神经元蜡样脂褐质沉积症家系神经元蜡样脂褐质沉积症（neuroaxonal dystrophy of adult life，NAD）是一种罕见的神经系统退行性变性疾病，主要累及中枢神经系统。

在中国，NAD的家系报告相对较少，因此，了解该疾病的病因和临床特征对于提高诊断和治疗水平是至关重要的。

经过长期的研究和临床观察，我们发现了一宗以癫痫起病的成人型NAD家系。

这个特殊家系中的患者常常在成年后发病，并出现了癫痫发作的症状。

我们对这家系的成员进行了详细的临床检查，并进行了基因突变分析。

基因突变分析结果显示，该家系的患者普遍存在DNAJC5基因突变。

DNAJC5基因编码的蛋白是一个重要的脑细胞内转运蛋白，对于细胞蜡样脂褐质（ceroid lipofuscin）的降解有重要作用。

该基因突变引起了DNAJC5蛋白功能异常，导致脑细胞内蜡样脂褐质的过度积累，最终导致神经元的损伤和功能障碍。

此外，我们还在家系成员的脑组织中进行了蜡样脂褐质沉积症的病理学研究。

研究结果显示，在患者的脑组织中观察到了显著增加的蜡样脂褐质沉积和神经元退变，这与成人型神经元蜡样脂褐质沉积症的典型病理特征一致。

针对该家系的临床特点和基因突变结果，我们建议家族成员进行基因检测，以早期发现潜在的患者并进行预防治疗。

此外，我们也呼吁加强对于NAD的病因和发病机制的研究，以提高诊断和治疗水平。

综上所述，通过对一宗以癫痫起病及DNAJC5基因突变的成人型神经元蜡样脂褐质沉积症家系的研究，我们了解了该疾病的临床特征和遗传基础。

这对于促进相关疾病的诊断和治疗具有重要意义。

希望未来能有更多的研究关注该疾病的病因和发病机制，以提供更好的临床管理和康复方案综合以上研究结果，我们发现DNAJC5基因突变是成人型神经元蜡样脂褐质沉积症的主要遗传基础。

该基因突变导致DNAJC5蛋白功能异常，进而导致脑细胞内蜡样脂褐质的过度积累，最终引起神经元损伤和功能障碍。

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FunctionalCharacterizationofNegriBodies(NBs)inRabiesVirus-InfectedCells:EvidencethatNBsAreSitesofViralTranscriptionandReplicationᰔ

XavierLahaye,1AuroreVidy,1†CarolePomier,1LindaObiang,1FrancisHarper,2YvesGaudin,1andDanielleBlondel1*

CNRS,UMR2472,INRA,UMR1157,IFR115,VirologieMole´culaireetStructurale,91198,GifsurYvette,France,1andCCNRS,FRE2937,IFR89,LaboratoiredeReplicationdel’ADNetUltrastructureduNoyau,94801Villejuif,France2

Received18March2009/Accepted26May2009RabiesvirusinfectioninducestheformationofcytoplasmicinclusionbodiesthatresembleNegribodiesfoundinthecytoplasmofsomeinfectednervecells.WehavestudiedthemorphogenesisandtheroleoftheseNegribody-likestructures(NBLs)duringviralinfection.Theresultsindicatethatthesesphericalstructures(oneortwopercellintheinitialstageofinfection),composedoftheviralNandPproteins,growduringtheviruscyclebeforeappearingassmallerstructuresatlatestagesofinfection.Wehaveshownthatthemicrotubulenetworkisnotnecessaryfortheformationoftheseinclusionbodiesbutisinvolvedintheirdynamics.Incontrast,theactinnetworkdoesnotplayanydetectableroleintheseprocesses.TheseinclusionbodiescontainHsp70andubiquitinylatedproteins,buttheyarenotmisfoldedproteinaggregates.NBLs,infact,appeartobefunctionalstructuresinvolvedinthevirallifecycle.Speciﬁcally,usinginsituﬂuorescenthybridizationtechniques,weshowthatallviralRNAs(genome,antigenome,andeverymRNA)arelocatedinsidetheinclusionbodies.Signiﬁcantly,short-termRNAlabelinginthepresenceofBrUTPstronglysuggeststhattheNBLsarethesiteswhereviraltranscriptionandreplicationtakeplace.

Viralinfectioncancauseextensivecellularrearrangementsleadingtotheformationofcytoplasmicinclusionsthatconcen-trateviralcomponents.Theseinclusionsareoftenconsideredasside-productsoftheinfectiousprocessduetothepassiveaccumulationoflargequantitiesofproteinsproducedinexcessduringinfection.Nevertheless,insomecases,ithasbeendem-onstratedthattheseinclusionsareinfactviralfactoriesinwhichessentialeventsoftheviralcycletakeplace(30,38).Indeed,inthesecompartments,thehighconcentrationoftheviralcomponentsincreasestheefﬁciencyofmanystepsoftheviralinfection(includingtranscriptionand/orreplicationofthegenomeandassemblyofsubviralparticles)suchthattheyrepresent“viralfactories.”Suchfactorieshavebeenidentiﬁedforavarietyofunrelatedviruses,includinglargeDNAviruses(suchasPoxviridae,Iridoviridae,andthecloselyrelatedAfricanswinefevervirus),smallDNAviruses(suchasadenovirusandpoliomavirus),double-strandedRNAviruses(suchasrotavi-ruses),andpositive-strandedRNAviruses(suchastogavirus)(39).Similarinclusionscalledaggresomesareformedinresponsetoproteinmisfolding(7,20).Proteinsaggregatesofmisfoldedproteinsaretoxictocellsandaredrivenalongmicrotubulestoaggresomesforimmobilizationandsubsequentdegradationbyproteasomes.Thesimilaritybetweenaggresomesandviralin-

clusionsraisesthepossibilitythatviruseshijacktheaggresomepathwaytofacilitatetheirownreplicationandassembly(14,39).Alternatively,aggresomesmaybepartoftheinnatecel-lularresponsethatrecognizesviralcomponentsasforeignormisfoldedandtargetsthemforstorageanddegradation.Rabiesvirus,theprototypeofthelyssavirusgenusthatbe-longstotheRhabdoviridaefamily(Mononegaviralesorder),causesafataldiseaseassociatedwithintenseviralreplicationinthecentralnervoussystem.Itssingle-strandednegative-senseRNAgenome(ϳ12kb),whichencodesﬁveviralpro-teins,isencapsidatedbythenucleoproteinN(50kDa)toformthenucleocapsidthatisassociatedwiththeRNA-dependentRNApolymeraseL(220kDa)anditscofactorthephospho-proteinP(33kDa).Insidetheviralparticle,thisnucleocapsidhasatightlycoiledhelicalstructurethatisassociatedwiththematrixproteinM(22kDa)andsurroundedbyamembranecontainingauniqueglycoproteinG(62kDa).Thevirusentersthehostcellthroughtheendosomaltransportpathwayviaalow-pH-inducedmembranefusionprocesscatalyzedbyglyco-proteinG(9).ThenucleocapsidreleasedintothecytoplasmservesasatemplatefortranscriptionandreplicationprocessesthatarecatalyzedbytheL-Ppolymerasecomplex.Duringtranscription,apositive-strandedleaderRNAandﬁvecappedandpolyadenylatedmRNAsaresynthesized.Thereplicationprocessyieldsnucleocapsidscontainingfull-lengthantigenome-senseRNA,whichinturnserveastemplatesforthesynthesisofgenome-senseRNA.Duringtheirsynthesis,boththenas-centantigenomeandthegenomeareencapsidatedbyNpro-teins.TheneosynthesizedgenomeeitherservesasatemplateforsecondarytranscriptionorisassembledwithMproteinstoallowbuddingoftheneosynthesizedvirionatacellularmem-brane.Someelectronmicroscopystudiessuggestthatthebud-