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石河子大学硕士学位论文非生物胁迫下棉花植物同源结构域（PHD）转录因子的研究学位申请人杨雷指导教师谢宗铭申请学位门类级别理学硕士学科、专业名称生物化学与分子生物学研究方向植物基因工程所在学院生命科学学院中国·新疆·石河子2013年6 月Characterization and Functional Analysis of Plant Homeodomain (PHD) Transcription Factors Responsiveto Abiotic Stress in CottonA Dissertation Submitted toShihezi UniversityIn Partial Fulfillment of the Requirementsfor the Degree of Master of Natural ScienceByYanglei(Biochemistry and Molecular Biology)Dissertation Supervisor:Prof. Xie ZongmingShihezi·Xinjiang·ChinaJun，2013学位论文独创性声明本人所呈交的学位论文是在我导师的指导下进行的研究工作及取得的研究成果。
据我所知，除文中已经注明引用的内容外，本论文不包含其他个人已经发表或撰写过的研究成果。
对本文的研究做出重要贡献的个人和集体，均已在文中作了明确的说明并表示谢意。
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研究生签名：时间：年月日导师签名：时间：年月日摘要目的：通过对陆地棉（Gossypium hirsutum L.）PHD（plant homeodomain）转录因子在棉花不同组织和非生物胁迫条件下的表达分析，筛选并克隆棉花逆境胁迫应答基因，初步探究棉花植物同源结构域转录因子在非生物下的应答模式以及转基因模式植物抗逆性能。
任文闯, 王欣, 张亚辉, 等. 玉米籽粒皱缩突变体sh2021的表型分析和基因定位[J]. 华南农业大学学报, 2023, 44(5): 750-759.REN Wenchuang, WANG Xin, ZHANG Yahui, et al. Morphological characterization and genetic mapping of shrunken endosperm mutant sh2021 in maize[J].Journal of South China Agricultural University, 2023, 44(5): 750-759.玉米籽粒皱缩突变体sh2021的表型分析和基因定位任文闯 ，王 欣，张亚辉，汤蕴琦，黄 君(华南农业大学 农学院, 广东 广州 510642)摘要: 【目的】分析玉米籽粒皱缩突变体的表型特征并进行籽粒相关基因的精细定位，为揭示该基因调控玉米籽粒发育的分子机制奠定基础。
【方法】以玉米自交系‘B73’种植过程中籽粒自发突变个体为材料，命名为shank2021(sh2021)，对其形态学和细胞学特征进行观察；构建分离群体，通过混合群体分离分析法(Bulked segregant analysis ，BSA)对基因进行初步定位，筛选交换单株进一步缩小定位区间，最后结合转录组测序及基因功能注释推测控制籽粒缺陷性状的候选基因。
【结果】与野生型相比，sh2021籽粒凹陷皱缩、颜色加深、籽粒排列不规则，且百粒质量降低。
扫描电镜观察发现，与野生型相比，sh2021胚乳细胞和淀粉粒均显著变小，且淀粉粒大小不均匀。
遗传分析结果表明，sh2021是由单个隐性基因突变所致。
利用BSA 分析方法将目的基因定位在3号染色体末端约13.25 Mb 区域。
进一步扩大分离群体筛选交换单株，将目的基因定位在标记ID5与I D 9之间的529.60 k b 范围。
研究论文李洪波，等：茯苓菌丝蛋白双向电泳体系的建立及质谱鉴定[5 ] RIOS J L. Chemical constituents and pharmacological properties of P oria cocos [J]. Planta Med ,2011,77(7)： 681-691.[6 ] CHEN X,ZHANG L,CHEUNG P C.Immunopotentiation and anti-tumor activity of carboxymethylated-sulfated beta- (l->3)-d-glucan from Poria cocos [J]. Int Immunopharmacol, 2010,10(4) ： 398-405.[7 ] CHANG H H,YEH C H,SHEU F. A novel immunomodulatory protein from Poria cocos induces toll-like receptor 4-dependentactivation within mouse peritoneal macrophages[J]. J Agric Food Chem,2009,57(14) $6129-6139.[8 ] CHANG H H, SHEU F. A Novel fungal immunomodulatory protein (PCP) isolated from Poria cocos activates mouse peritonealmacrophage involved in Toll-like receptor 4[J]. FASEB J ,2007,21 (702) ： 15.[9 ] WU Zongxun,SUN Zhiwen. Construction and analysis of EST library in Wolfiporia cocos [J]. Bio Formosa, 2007,42 (1) ： 47-53.(in Chinese)[10] LI Hongbo,YANG Ruixue,CHEN Jing,et al. Preparation and analysis of the antibody against Poria cocos immunomodulatoryprotein WCFIP1[J]. Chinese Traditional and Herbal Drugs,2014,45(20)：2912-2916.(in Chinese)[11] HOU Y,ZHENG Z,XU S,et al. Proteomic analysis of F usarium graminearum treated by the fungicide JS399-19[J]. Pestic BiochemPhysiol,2013,107(1)：86-92[12] SU Y,GUO Q,TU J,et al. Proteins differentially expressed in conidia and mycelia of the entomopathogenic fungus Metarhiziumanisopliae sensu stricto[J]. Can J Microbiol ,2013,59(7) ： 443-448.[13] QIU J,SU Y,GELBIC I,et al. Proteomic analysis of proteins differentially expressed in conidia and mycelium of theentomopathogenic fungus Aschersonia placenta[J].Can J Microbiol ,2012,58(12)：1327-34.[14] LI Yi,YANG Sheng,LI Chen,et al. Compound medicinal medium of flask liquid fermentation of Poria cocos and its chemicalconstituents[J]. Chinese Traditional and Herbal Drugs,2012,43(8)： 1519-1522.(in Chinese)科技信息欧盟拟禁止在食品中使用山梨酸钙2017年11月17日，据《食品导航》网站消息，由于缺乏山梨酸钙防腐剂的使用安全性数据，欧盟将在食品中禁止 使用山梨酸钙。
㊃病例报告㊃小型无绿藻致血流感染1例及文献复习张旭李晨潘娟李足意聂格(浏阳市中医医院,浏阳410300)ʌ摘要ɔ患者,男,44岁,农民㊂全身皮肤散在红色斑疹㊁左小腿皮肤溃烂伴发热㊂在皮肤伤口分泌物及血液中均培养出酵母样菌落,菌落涂片显示不同大小的球形孢子囊,具有桑葚样外观㊂最终确诊为无绿藻菌感染,予以氟康唑㊁伊曲康唑治疗后死亡㊂本病例包含的数据及其文献复习将增加医务工作者对该病原体的认识㊂ʌ关键词ɔ小型无绿藻菌;血流感染;皮肤感染ʌ中图分类号ɔ R519.5ʌ文献标志码ɔ A ʌ文章编号ɔ1673-3827(2023)18-0246-04B l o o d s t r e a m i n f e c t i o n c a u s e d b y P r o t o t h e c a w i c k e r h a m i i:a c a s e r e p o r t a n d r e v i e w o f t h e l i t e r a t u r e sZ HA N G X u,L I C h e n,P A N J u a n,L I Z u y i,N I E G e(L i u y a n g H o s p i t a l o f T r a d i t i o n a l C h i n e s e M e d i c i n e,L i u y a n g H u n a n410300,C h i n a)ʌA b s t r a c tɔ A44-y e a r-o l d m a l e f a r m e r p r e s e n t e d w i t h e r y t h e m a t o u s p l a q u e s o f t h e w h o l e b o d y,d i f f u s e l y i n f i l t r a t e d e r o s i o n s o n h i s l e f t l e g a n d f e v e r.S m o o t h,c r e a m y-w h i t e,y e a s t-l i k e c o l o n i e s g r e w f r o m s k i n b i o p s y s p e c i m e n a n d b l o o d s p e c i m e n a f-t e r72h o f i n c u b a t i o n.E l e c t r o n m i c r o s c o p y d e m o n s t r a t e d m o r u l a o r d a i s y-l i k e a p p e a r a n c e o f i t s e n d o s p o r u l a t i n g s p o r a n g-i a.T h e s e f i n d i n g s w e r e c o n s i s t e n t w i t h a d i a g n o s i s o f p r o t o t h e c o s i s.T h e p a t i e n t w a s t r e a t e d w i t h f l u c o n a z o l e a n d i t r a c o n-a z o l e b u t f i n a l l y d i e d.T h i s c a s e a n d t h e l i t e r a t u r e r e v i e w w i l l i n c r e a s e t h e k n o w l e d g e o f t h i s p a t h o g e n.ʌK e y w o r d sɔP r o t o t h e c a w i c k e r h a m i i;b l o o d s t r e a m i n f e c t i o n;c u t a n e o u s p r o t o t h e c o s i s[C h i n J M y c o l,2023,18(3):246-249]无绿藻是一种普遍存在于自然界的条件性致病真菌㊂该菌属于绿藻门㊁T r e b o u x l o p h y c a e纲㊁绿藻目㊁绿藻科㊁无绿藻属[1]㊂迄今为止,已鉴定出16种无绿藻[2-3],包括大型无绿藻(P.s t a g n o r a)㊁中型无绿藻(P.z o p f i i)㊁小型无绿藻(P.w i c k e r-h a m i i)㊁P.b l a s c h k e a e㊁P.c u t i s㊁P.m i y a j i i㊁P. t u m u l i c o l a㊁P.c i f e r r i i㊁P.b o v i s㊁P.u l m e a㊁P. m o r i f o r m i s㊁P.c e r a s i㊁P.c o o k e i㊁P.p r i n g s h e-i m i i㊁P.x a n t h o r i a e和P.p a r a c u t i s㊂目前已证实中型无绿藻㊁小型无绿藻㊁P.b l a s c h k e a e㊁P.c u t i s㊁P.m i y a j i i与人类无绿藻病相关[4]㊂本文报告了1例由皮肤无绿藻病发展为血流感染的病例,并汇总了截止于2022年8月份全球无绿藻血流感染病例㊂作者简介:张旭,女(汉族),硕士,主管药师.E-m a i l:v i c k e y z x@ 163.c o m 1病例资料患者,男,44岁㊂因阵发性喘息㊁气促20余年伴发左小腿皮肤溃烂10余天,发热1d入院㊂患者1年前无明显诱因,全身皮肤出现散在的红色斑疹,间有瘙痒㊂2020年2月诊断为湿疹,予以卤米松治疗㊂1个月前,患者皮疹面积变大且伴有溃疡,痰标本检出诺卡菌㊂曾予以亚胺培南西司他汀联合复方磺胺甲恶唑治疗后肺部症状好转㊂10余天前出现左小腿皮肤溃疡㊂患者既往有哮喘及糖尿病,近半年自行口服地塞米松片(4~8m g/d)㊂体格检查入院体温为38.9ħ㊂双肺可闻及干湿性啰音,全身皮肤可见散在的红色斑疹,间有瘙痒,双下肢中度浮肿,左小腿皮肤溃烂,大小约4 c mˑ7c m左右,伴少许黄色渗液(见图1)㊂辅助检查胸部C T示:左上肺及双下肺多发病灶,性质待定㊂相关感染指标:白细胞:12.71ˑ㊃642㊃中国真菌学杂志2023年6月第18卷第3期 C h i n J M y c o l,J u n e2023,V o l18,N o.3Copyright©博看网. All Rights Reserved.109/L ,中性粒细胞比率:97.6%,C -反应蛋白:205.20m g /L ,降钙素原:3.4n g/m L ,G 试验:291.51μg /L ,GM 试验:0.75μg/L ㊂微生物检查 将患者皮损采集的标本以及血培养阳性的标本接种于沙堡弱琼脂培养基和念珠菌显色培养基中,在沙堡弱培养基中的菌落为白色的酵母样菌落(见图2),而在显色培养基中的菌落随着时间的延长由粉色变成蓝色(见图3)㊂并对该菌进行革兰染色(见图4)以及湿片观察形态(见图5),最终通过生物梅里埃公司V i t e k 2C o m p a c t 自动化系统(Y S T 检测显示:生物编码为4502100000205130)与MA L D I -T O F -M S 鉴定其为小型无绿藻㊂诊断 根据患者临床症状及真菌检测结果诊断为小型无绿藻所致皮肤及血流感染㊂治疗 初始经验性予以亚胺培南西司他汀㊁复方磺胺甲恶唑联合氟康唑注射液治疗㊂由于医疗费用问题,家属拒绝使用两性霉素B ㊂治疗3d 后予以停用氟康唑注射液,改用伊曲康唑(200m g/d)口服㊂出院带药:伊曲康唑㊁复方磺胺甲恶唑片和米诺环素㊂出院约3周后,患者死亡㊂由于未进行无绿藻药敏试验,且治疗疗程较短易导致复发及转移,该患者的死亡不能排除无绿藻血流感染控制不佳所导致的影响㊂图1 左腿软组织感染伤口 图2 沙堡弱琼脂培养基中菌落形态(35ħ,72h ) 图3 念珠菌显色培养基中菌落形态(35ħ,72h)F i g .1 D i f f u s e l y i n f i l t r a t e d e r o s i o n s o n h i s l e f t l e g F i g .2 T h e c o l o n y m o r p h o l o g y o n t h e S D A m e d i u m (35ħ,72h o u r s ) F i g.3 T h e c o l -o n y m o r p h o l o g y o n t h e o n C H R OM a ga r C a n d i d a (35ħ,72h)图4 阳性血瓶直接革兰染色 图5 菌落生理盐水湿片染色显示桑椹状孢子囊F i g .4G r a m s t a i n i n g o f p o s i t i v e b l o o d s a m p l e F i g.5 U n s t a i n e d m u l b e r r y -l i k e s p o r a n gi a 2 文献回顾以 P r o t o t h e c a ㊁pr o t o t h e c o s i s 为关键词,在中国知网㊁万方数据库㊁P u b m e d 数据库中进行检索㊂截止2022年,包括本例在内,无绿藻血流感染病例共有21例[5-24](见表1),其中男性15例,女性6例,平均年龄50岁㊂除1例未明确到种外,其余14例为小型无绿藻㊁6例为中型无绿藻㊂值得注意的是,无绿藻血流感染死亡率较高为62%,约81%的无绿藻血流感染者免疫功能受到抑制,推测死亡率与患者自身免疫力具有一定相关性㊂此外,62%血流感染者伴有皮肤感染,由皮肤播散到血液可能是其主要感染途径㊂3 讨 论无绿藻感染虽然较罕见,但随着微生物培养技术及分子生物学方法的提高,其检出率在近10年呈上升趋势[25-26]㊂迄今为止,全世界已有约200多例无绿藻感染病例[27]㊂由中国报道的无绿藻感染㊃742㊃ 中国真菌学杂志2023年6月第18卷第3期 C h i n J M yc o l ,J u n e 2023,V o l 18,N o .3 Copyright ©博看网. All Rights Reserved.表1无绿藻血流感染病例文献报道T a b.1 C a s e s o f b l o o d s t r e a m i n f e c t i o n d u e t o p r o t o t h e c o s i s序列[引用]发表时间地区年龄/性别菌种免疫状态危险因素感染部位治疗转归1[5]1974新西兰29/M P w正常无皮肤㊁血液㊁肝脏AM B治愈2[6]1991南非7/M P w抑制MO P P治疗方案㊁希克曼静脉导管血液AM B治愈3[7]1996美国59/F P z抑制肺移植血液不详死亡4[8]1997美国75/M P w抑制使用免疫抑制剂血液㊁皮肤AM B治愈5[9]2002美国19/M不详抑制急性白血病㊁服用大剂量激素血液AM B不详6[10]2004美国56/M P w抑制干细胞移植㊁免疫抑制治疗血液㊁皮肤AM B死亡7[11]2004美国49/M P w抑制艾滋病㊁外伤血液㊁皮肤㊁AM B死亡8[12]2004澳大利亚58/M P z抑制干细胞移植㊁环孢霉素血液㊁皮肤㊁肺㊁心㊁肾㊁肝脏AM B死亡9[13]2005美国58/M P w抑制干细胞移植㊁免疫抑制治疗血液㊁皮肤㊁中枢AM B死亡10[14]2008美国61/M P w抑制肝移植㊁糖尿病㊁免疫抑制治疗血液㊁皮肤M I F㊁AM B死亡11[15]2010英国49/F P w抑制白血病血液㊁皮肤AM B㊁V R C好转12[16]2011澳大利亚78/F P w抑制心脏移植㊁糖尿病㊁免疫抑制治疗血液㊁皮肤AM B㊁I T C死亡13[17]2011马来西亚61/F P w不详肾移植血液无死亡14[18]2014中国85/M P w不详使用泼尼松2年皮肤㊁血液K T Z好转15[19]2015日本65/F P w抑制系统性红斑狼疮㊁糖尿病㊁口服甲泼尼龙血液㊁皮肤AM B㊁V R C好转16[20]2015美国59/M P w抑制肾移植㊁糖尿病血液V R C㊁AM B死亡17[21]2018印度36/M P z抑制肝移植㊁免疫抑制治疗血液㊁皮肤㊁肺AM B死亡18[22]2019阿根廷19/M P z抑制白血病㊁免疫抑制治疗血液AM B治愈19[23]2019中国13/M P z抑制白血病血流F L C死亡20[24]2021德国61/F P z抑制白血病㊁糖尿病血液㊁肺㊁肝脏㊁脑AM B死亡本例2022中国44/F P w正常糖尿病㊁口服地塞米松㊁低蛋白血症血液㊁皮肤F L C㊁I T C死亡F:女;M:男;P w:小型无绿藻;P z:中型无绿藻;AM B:两性霉素B;M I F:米卡芬净;V R C:伏立康唑;I T C:伊曲康唑;K T Z:酮康唑;F L C:氟康唑㊂病例约为29例[28-34],本例属于湖南省首次报道的无绿藻感染病例且为中国第2例小型无绿藻血流感染病例㊂无绿藻所致感染主要表现为皮肤感染㊁滑膜炎及其纤维组织炎和播散性感染[26]㊂中国报道病例中85%为皮肤感染㊂皮肤型的无绿藻病通常预后良好,死亡率只有1%㊂但其播散型的死亡率高达56%[27]㊂其结果与本文统计的血流感染死亡率相近㊂菌种的毒力是否与无绿藻病死亡率相关,目前这种关系尚不明确㊂但有研究表明[1,36],与小型无绿藻相比,中型无绿藻对免疫抑制小鼠更具有致死性㊂其次无绿藻所致播散性感染与患者免疫力相关㊂据统计播散性感染患者中约89.2%伴有免疫功能抑制,其在器官及干细胞移植者中最为常见㊁白血病患者次之[37]㊂随着全球免疫功能低下个体数量的增加,由无绿藻引起的播散性感染的发生率必然会增加㊂但由于缺乏对该病原体的认识,其最初的皮肤感染症状常常被延误治疗㊂现将无绿藻皮肤病变的特征汇总如下[28],以便临床鉴别:无绿藻皮肤感染病变通常呈多态性,四肢和面部是感染的主要部位;红斑丘疹和斑块是最常见的皮肤表现;红斑病变可伴有浅表溃疡㊁坏死结痂㊁脓疱或大疱;有些病变类似湿疹,但无绿藻皮肤病变多呈不对称分布㊂目前美国临床和实验标准化协会尚未对无绿藻体外药敏试验制定出相应的标准,部分实验室参照酵母药敏试验C L S I M27-A3文件进行结果判断㊂体外药敏试验显示:无绿藻对两性霉素B最为㊃842㊃中国真菌学杂志2023年6月第18卷第3期 C h i n J M y c o l,J u n e2023,V o l18,N o.3Copyright©博看网. All Rights Reserved.敏感,伏立康唑次之[28,31-34]㊂中国报道病例中,暂未发现对两性霉素B耐药的菌株,但无绿藻对棘白菌素类耐药,原因可能为无绿藻的细胞壁中缺乏1,3-β-D-葡聚糖[38]㊂此外,1例药敏试验显示[29]:无绿藻对米诺环素敏感㊂这似乎与无绿藻为真菌相矛盾,但有研究显示[39]:无绿藻既有真核生物的基因又有细菌的基因㊂推测无绿藻是由一种含28S r R N A基因的类真核生物吞噬了一种含16S r R N A 基因的类蓝细菌之后,共同演化而成㊂对于无绿藻感染,国内外暂无标准的治疗方案㊂病例报道中多使用多烯及三唑类药物治疗㊂除药物治疗外,外科手术被认为是治疗局部无绿藻感染的另一种有效的方法㊂本例患者经氟康唑与伊曲康唑治疗后死亡㊂这种罕见的病原体对人类健康造成的威胁正在日益增加㊂临床医生应警惕无绿藻由局部感染发展为全身感染的趋势㊂及时的诊断和治疗对预后具有重要意义㊂参考文献[1]L A S S-F LÖR L C,MA Y R A.H u m a n p r o t o t h e c o s i s[J].C l i nM i c r o b i o l R e v,2007,20(2):230-242.[2]K A N O R,K A Z U O S,Y A G U C H I T,e t a l.P h e n o t y p i c c h a r-a c t e r i s t i c s o f P r o t o t h e c a s p e c i e s o c c u r r i n g i n h u m a n s a n d a n-i m a l s[J].M e d M y c o l,2022,63(1):17-20.[3]J A G I E L S K I T,B A K U L A Z,G AWO R J,e t a l.T h e g e n u sP r o t o t h e c a(T r e b o u x i o p h y c e a e,C h l o r o p h y t a)r e v i s i t e d:I m-p l i c a t i o n s f r o m m o l e c u l a r t a x o n o m i c s t u d i e s[J].A l g a l R e-s e a r c h,2019,43(7):1-19.[4]N I S H I MU R A K,MA S U D A M.M o l e c u l a r c h a r a c t e r i z a t i o no f P r o t o t h e c a s t r a i n s i s o l a t e d i n C h i n a r e v e a l e d t h e f i r s t c a-s e s o f p r o t o t h e c o s i s a s s o c i a t e d w i t h P r o t o t h e c a z o p f i i g e n o-t y p e1[J].M e d M y c o l,2018,56(3):279-287. 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All Rights Reserved.本例患者一经确诊,立即采用光动力疗法[11-12]联合抗真菌药物治疗,光动力疗法能使细胞壁通透性升高从而降低一些抗生素的最低抑菌浓度(M I C),即增强抗真菌药物的疗效,促进皮损恢复,降低复发率;伊曲康唑对致病性暗色真菌有较强的抑制作用,已逐渐取代其他药物而成为治疗着色芽生菌病的首选药物,口服200~400m g,连续3 ~12个月[13-14]㊂本例患者口服伊曲康唑㊁涂抹药物和光动力疗法联合应用,临床效果较好㊂参考文献[1]Q U E I R O Z-T E L L E S F,HO O G S D,S A N T O S D,e t a l.C h r o m o b l a s t o m y c o s i s[J].C l i n M i c r o b i o l R e v,2017,30(1):233-276.[2]赵作涛,鲁巧云,高那,等.首次报道中国北方F o n s e c a e am o n o p h o r a所致着色芽生菌病1例及其相关实验研究[J].中国真菌学杂志,2010,5(4):3.[3]秦琴,晏文,鲁东平,等.F o n s e c a e a m o n o p h o r a所致着色芽生菌病[J].临床皮肤科杂志,2017,46(12):4.[4]X I L Y,S U N J F,L U C M,e t a l.M o l e c u l a r d i v e r s i t y o fF o n s e c a e a(C h a e t o t h y r i a l e s)c a u s i n g c h r o m o b l a s t o m y c o s i si n s o u t h e r n C h i n a[J].M e d M y c o l,2009,47(1):27-33.[5]D E B,S E M B L A N O B.C h r o m o b l a s t o m y c o s i s:a n e t i o l o g i-c a l,e p ide m i o l o g i c a l,c l i n i c a l,d i a g n o s t i c,a n d t r e a t m e n t u p-d a t e[J].A n B r a s De r m a t o l,2018,93(4):495-506.[6]孙祺琳,余敏,陈骏,等.中国大陆地区52例皮肤着色芽生菌病回顾性分析[J].中国真菌学杂志,2020,15(2):5.[7]D E H O O G G S,A T T I L I-A N G E L I S D,V I C E N T E V A,e ta l.M o l e c u l a r e c o l o g y a n d p a t h o g e n i c p o t e n t i a l o f F o n s e c a e as p e c i e s[J].M e d M y c o l,42(5):405-416.[8]王丽,王薇.F o n s e c a e a m o n o p h o r a色素毒力性研究[J].中国真菌学杂志,2021,16(2):77-83.[9]张信江,鲁东平.皮肤性病基层医师诊疗手册[M].人民卫生出版社,2014.[10] AM E E N M.M a n a g i n g c h r o m o b l a s t o m y c o s i s[J].T r o p i c a lD o c t o r,2010,40(2):65-67.[11]兰文雷,陈忠业,王丽金,等.5-氨基酮戊酸光动力联合伊曲康唑治疗着色芽生菌病1例[J].中国真菌学杂志,2015, 10(4):3.[12] HU Y X,Q I X Y,S U N H B,e t a l.P h o t o d y n a m i c t h e r a p yc o m b i n ed w i t h a n t i f u n g a l d r u g s a g a i n s t c h r o m o b l a s t o m y c o-s i s a n d t h e e f f e c t o f A L A-P D T o n F o n s e c a e a i n v i t r o[J].P L o S N e g l T r o p D i s,2019,13(10):e0007849. 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All Rights Reserved.。
不对称自由基反应英文Asymmetric Radical Reactions: An Insight into Their Mechanism and Applications.Introduction.Asymmetric radical reactions have emerged as a powerful tool in organic synthesis, enabling the synthesis of chiral compounds with high enantiomeric purity. These reactions differ significantly from their symmetric counterparts, as they involve the generation and utilization of chiral radicals. These chiral radicals can undergo a range of reactions, including substitution, addition, and cyclization, leading to the formation of enantiomerically enriched products.Mechanism of Asymmetric Radical Reactions.The mechanism of asymmetric radical reactions typically involves three key steps: radical generation, chiralitytransfer, and radical termination.Radical Generation.The first step involves the generation of a radical species. This can be achieved through various methods, such as photolysis, thermal decomposition, or redox reactions. The generated radical can be chiral or achiral, depending on the starting materials and the conditions used.Chirality Transfer.The second step involves the transfer of chirality from a chiral auxiliary or catalyst to the radical species. This chirality transfer can occur through covalent or non-covalent interactions between the catalyst/auxiliary and the radical. The nature of these interactions determines the stereoselectivity of the reaction.Radical Termination.The final step involves the termination of the radicalspecies, leading to the formation of the desired product. This termination can occur through various mechanisms, such as coupling with another radical species, hydrogen atom abstraction, or disproportionation.Applications of Asymmetric Radical Reactions.Asymmetric radical reactions have found widespread applications in various fields of organic synthesis, including the synthesis of natural products, pharmaceuticals, and functional materials.Synthesis of Natural Products.Natural products often possess complex chiral structures, making their synthesis challenging. Asymmetric radical reactions have proven to be effective tools for the synthesis of such chiral natural products. For example, the use of chiral radicals generated from appropriate precursors has enabled the enantioselective synthesis of alkaloids, terpenes, and amino acids.Pharmaceutical Applications.The enantiomers of chiral drugs often differ significantly in their biological activities, making it crucial to control their enantiomeric purity. Asymmetric radical reactions can be used to synthesize enantiomerically enriched chiral drugs with high selectivity. This approach has been successfully applied to the synthesis of various drugs, including anti-inflammatory agents, anticancer agents, and antiviral agents.Functional Materials.Chiral materials possess unique physical and chemical properties that make them useful in various applications, such as displays, sensors, and catalysts. Asymmetricradical reactions can be used to synthesize chiral building blocks for the preparation of such materials. For instance, chiral polymers can be synthesized by utilizing asymmetric radical polymerization reactions, leading to the formation of materials with controlled chirality and tailored properties.Conclusion.Asymmetric radical reactions have emerged as powerful tools for the synthesis of enantiomerically enriched chiral compounds. Their unique mechanism, involving chirality transfer from a chiral catalyst/auxiliary to the radical species, enables high selectivity and enantiopurity in the product. The widespread applications of asymmetric radical reactions in organic synthesis, particularly in the synthesis of natural products, pharmaceuticals, and functional materials, highlight their importance in modern chemistry.Future Perspectives.Despite the significant progress made in the field of asymmetric radical reactions, there are still numerous challenges and opportunities for further exploration.Improving Selectivity and Efficiency.One of the key challenges in asymmetric radical reactions is achieving high selectivity and efficiency. While significant progress has been made in this area, there is still room for improvement. Future research could focus on developing new chiral catalysts/auxiliaries that can promote asymmetric radical reactions with higher selectivity and efficiency.Expanding the Scope of Reactions.Currently, the scope of asymmetric radical reactions is limited by the availability of suitable precursors and the reactivity of the generated radicals. Future research could aim to expand the scope of these reactions by developing new methods for generating radicals with desired functionalities and reactivities.Applications in Sustainable Chemistry.In the context of sustainable chemistry, asymmetric radical reactions offer an attractive alternative to traditional synthetic methods. By utilizing renewableresources and mild reaction conditions, asymmetric radical reactions could contribute to the development of more sustainable synthetic routes for the preparation of chiral compounds.Integration with Other Techniques.The integration of asymmetric radical reactions with other techniques, such as photocatalysis, electrochemistry, and microfluidics, could lead to the development of new and innovative synthetic methods. By combining the advantages of these techniques, it may be possible to achieve even higher selectivity, efficiency, and scalability in asymmetric radical reactions.In conclusion, asymmetric radical reactions have emerged as powerful tools for the synthesis of enantiomerically enriched chiral compounds. While significant progress has been made in this area, there are still numerous opportunities for further exploration and development. Future research in this field could lead tothe discovery of new and innovative synthetic methods with improved selectivity, efficiency, and sustainability.。
Phenotypic Characterization of DisseminatedCells with TSC2Loss of Heterozygosity inPatients with LymphangioleiomyomatosisXiong Cai1,Gustavo Pacheco-Rodriguez1,Qing-Yuan Fan1*,Mary Haughey1,Leigh Samsel2,Souheil El-Chemaly1, Hai-Ping Wu1,J.Philip McCoy2,Wendy K.Steagall1,Jing-Ping Lin3,Thomas N.Darling4,and Joel Moss11Cardiovascular and Pulmonary Branch,2Flow Cytometry Core Facility,and3Office of Biostatistics Research,Division of Cardiovascular Science, National Heart,Lung,and Blood Institute,National Institutes of Health,Bethesda,Maryland;and4Department of Dermatology,Uniformed Services University of the Health Sciences,Bethesda,MarylandRationale:Lymphangioleiomyomatosis(LAM),occurring sporadi-cally(S-LAM)or in patients with tuberous sclerosis complex(TSC),results from abnormal proliferation of LAM cells exhibiting muta-tions or loss of heterozygosity(LOH)of the TSC genes,TSC1or TSC2.Objectives:To identify molecular markers useful for isolating LAMcells from bodyfluids and determine the frequency of TSC1or TSC2LOH.Methods:Candidate cell surface markers were identified using genemicroarray analysis of human TSC22/2cells.Cells from bronchoal-veolar lavagefluid(BALF),urine,chylous effusions,and blood weresorted based on reactivity with antibodies against these proteins(e.g.,CD9,CD44v6)and analyzed for LOH using TSC1-and TSC2-related microsatellite markers and single nucleotide polymorphismsin the TSC2gene.Measurements and Main Results:CD44v61CD91cells from BALF,urine,and chyle showed TSC2LOH in80%,69%,and50%of patientsamples,M cells with TSC2LOH were detected inmore than90%of blood M cells from different bodyfluids of the same patients showed,in most cases,identical LOHpatterns,that is,loss of alleles at the same microsatellite loci.In a fewpatients with S-LAM,LAM cells from different bodyfluids differed inLOH patterns.No patients with S-LAM with TSC1LOH were identi-fied,suggesting that TSC2abnormalities are responsible for the vastmajority of S-LAM cases and that TSC1-disease may be subclinical.Conclusions:Our data support a common genetic origin of LAM cellsin most patients with S-LAM,consistent with a metastatic model.Insome cases,however,there was evidence for genetic heterogeneitybetween LAM cells in different sites or within a site.Keywords:lymphangioleiomyomatosis;metastasis;loss of heterozy-gosity;CD9;CD44v6Lymphangioleiomyomatosis(LAM)is a rare multisystem disease affecting primarily women,characterized by abnormal prolifer-ation of smooth muscle–like LAM cells,which leads to cystic destruction of the lungs,formation offluid-filled cystic structures in the axial lymphatics(e.g.,lymphangioleiomyomas),and renal angiomyolipomas(AMLs)(1–5).LAM occurs as a sporadic disease(S-LAM)or in association with tuberous sclerosis com-plex(TSC)(6–8).TSC is an autosomal dominant syndrome characterized by multiorgan hamartomas,resulting from muta-tions in one of two tumor suppressor genes,TSC1on chromosome 9(9q34)(9)and TSC2on chromosome16(16p13.3)(10,11). LAM cells in S-LAM were reported to be associated with TSC2 loss of heterozygosity(LOH)(12–15),consistent with Knudson’s ‘‘two-hit’’hypothesis(16).LAM cells from lung nodules,AMLs,and lymph nodes of the same patient showed identical TSC2mutations and LOH pat-terns(13–15),consistent with metastatic spread among organs. Further supporting this model,LAM cells were identified in donor lungs after transplantation(17,18)and could be isolated from blood,urine,and chyle of patients with LAM(19,20), consistent with LAM cell dissemination in bodyfluids.Identifi-cation of LAM cells in blood by LOH was aided byfluorescence-activated cell sorting(FACS)removal of non-LAM cells after immunostaining with antibodies against leukocyte common antigen(CD45)and glycophorin A(CD235a)(19),a protein present on LAM cells in lung nodules.In our previous study(19), we were able to isolate LAM cells from only approximately60% of patients,and thus could not answer questions such as whether sporadic LAM was primarily TSC2driven,whether LAM cells in different bodyfluids showed similar LOH patterns,orwhether(Received in original form March20,2010;accepted infinal form July16,2010) *Current affiliation:Department of Neuroscience,Cleveland Clinic,Cleveland, Ohio.Supported in part by the Intramural Research Program of the National Institutes of Health,National Heart,Lung,and Blood Institute(J.M.);and by RO1 CA100907(T.N.D.).Correspondence and requests for reprints should be addressed to Joel Moss, M.D.,Ph.D.,Translational Medicine Branch,National Heart,Lung,and Blood Institute,National Institutes of Health,Building10,Room6D05,MSC-1590, Bethesda,MD20892-1590.E-mail:mossj@This article has an online supplement,which is accessible from this issue’s table of contents at Am J Respir Crit Care Med Vol182.pp1410–1418,2010Originally Published in Press as DOI:10.1164/rccm.201003-0489OC on July16,2010 Internet address:LAM cells could be isolated from bronchoalveolar lavagefluid (BALF).These questions prompted us to identify cell surface molecules unique to TSC22/2cells and use thesefindings to isolate LAM cells with TSC2LOH from BALF,urine,chyle,and blood.We have shown that CD44v6is expressed in situ by LAM cells in lung nodules and is present on LAM cells grown from explanted lungs (21).This splice variant of the hyaluronic acid receptor is believed to be involved in tumor metastasis and progression(22–24).In the present study,we showed that the tetraspanin CD9,a highly expressed gene identified by microarray analysis of TSC22/2cells from TSC skin lesions(25),and CD44v6identified LAM cells with TSC2LOH from BALF,urine,and chylous effusions. Similarly,CD452CD235a2and CD452CD235a1cells with TSC2 LOH were detected in blood cell fractions.The majority of TSC2 LOH patterns were identical in LAM cells from blood,urine,and BALF or chyle from the same patients.Different LOH patterns, however,were identified in LAM cells from different bodyfluids in a minority of patients with S-LAM.Furthermore,we failed to find TSC1LOH in patients with S-LAM.Some of the results of these studies have been previously reported in the form of an abstract(26).METHODSSupplemental description of methods is available in the online supplement.Patients and Sample CollectionSamples were collected from randomly selected patients with LAM(45 S-LAM and10TSC-LAM)and13healthy female volunteers who were enrolled between2007and2009at the National Institutes of Health Clinical Center in clinical protocols(95-H-0186,96-H-0100)approved by the National Heart,Lung,and Blood Institute Institutional Review Board.The diagnosis of LAM was based on clinical,radiologic,and/or histopathologicfindings.Isolation of Cells from TSC Skin BiopsiesFibroblasts(TSC21/2)from postauricular normal-appearing skin and fibroblast-like cells(TSC22/2)from periungualfibromas of toes from the same female patients with TSC enrolled in protocol00-H-0051 were isolated and grown as described(25).Immunofluorescence Analysis of Cultured Cellsby Confocal MicroscopyAs reported,cells were incubated with a mouse monoclonal antibody against CD9(1:20;BD Biosciences,San Jose,CA)at48C for approx-imately12hours and then with the immunofluorescentfluorescein isothiocyanate(FITC)-labeled goat antibody against mouse IgG(1:100 dilution;Vector Laboratories,Burlingame,CA)for1hour at room temperature(21).Fluorescence-activated Cell SortingAnti–CD44v6-FITC(clone VFF-7)and anti–CD9-R-phycoerythrin(PE) (clone MM2/57)antibodies were purchased from Invitrogen(Carlsbad, CA).Anti–CD9-FITC(clone M-L13),anti–CD44-R-PE(clone G44-26), anti–CD45-FITC(clone HI30),and anti–CD235a-PE(clone GA-R2) antibodies were from BD Biosciences.Cells from blood,urine,BALF, and chylous effusions were labeled forflow cytometric analysis and sorting by incubation for30minutes at room temperature with the indicated antibodies,followed by two washes with PBS and sorting in a MoFlo Flow Cytometer(Beckman Coulter,Inc.,Fullerton,CA). Fluorescence signals were collected using amplifiers that reported on a logarithmic scale.Data acquisition,analysis,and compensation were performed using Summit software(Beckman Coulter).Polymerase Chain Reaction Analysis of LOHGenomic DNA was isolated from whole blood and unsorted or sorted cells using the QIAamp DNA Micro Kit(QIAGEN,Valencia,CA)and amplified(19)to determine LOH.Briefly,genomic DNA se-quences were amplified at loci D16S291,Kg8,D16S3395,D16S3024, and D16S521on chromosome16p13.3,and at loci D9S149,D9S1198, and D9S66on chromosome9q34.Primer sequences were obtained from the UniSTS Database(/unists).Antisense primers were labeled with6-FAM(Invitrogen).Polymerase chain reaction(PCR)products were analyzed on a3100Genetic Analyzer (Applied Biosystems,Foster City,CA).Q LOH was calculated as described(19).Q LOH values of less than0.5or more than0.62were scored as LOH or retention of heterozygosity(ROH),respectively, whereas no definite decision was made with Q LOH values of0.5to0.62 (19,27).Single-Nucleotide Polymorphism AnalysisGenomic DNA isolated from whole blood and unsorted or sorted cells was amplified by PCR for the exon40polymorphism(T5202C;rs1748) (28,29)or for a splice site polymorphism(C482–3T;rs1800720)(28, 30),using these primer sequences:TSC40S-Hex,59-Hex-ATGGAG GGCCTTGTGGACAC-39,and TSC40AS,59-CGGAGCCGCTTGA TGTG-39;TSCspliceS,59-GGAGATGTAGATTCGGCGTC-39,and TSCspliceAS-Hex,59-Hex-CTGCGGAGCTGAACTTAGG-39.PCR products were digested with the appropriate restriction enzyme (EcoRV for T5151C;PvuII for C482–3T)(New England Biolabs, Beverly,MA),and then analyzed with a3100Genetic Analyzer (Applied Biosystems).Statistical AnalysisFisher exact test was performed with the SPSS15.0(SPSS,Inc., Chicago,IL).Statistical significance was accepted for P,0.05. RESULTSWe hadfirst detected LAM cells with TSC2LOH in blood by OncoQuick density-gradient fractionation,and from urine and chyle specimens based on centrifugation.To improve yield and purity of LAM cells we focused on identification of potential LAM cell surface markers by comparing gene expression in TSCfibroblasts(TSC21/2)grown from normal-appearing skin and infibroblastic cells(TSC22/2)grown from TSC-associated skin tumors of the same patient.By microarray analysis,levels of CD9were higher in TSC22/2cells than in their TSC21/2 counterparts(25).We reported previously that LAM cells grown from lungs contained CD44v6,a splice variant of the hyaluronic acid receptor CD44(21),prompting us to use antibodies to these proteins to isolate circulating LAM cells. CD9Expression on TSC22/2CellsAmounts of CD9assessed byflow cytometric analysis and immunostaining were greater in TSC22/2skin tumor cells than in TSC21/2skinfibroblasts(Figure1).As determined by mean fluorescence intensity(MFI),the levels of CD9were much higher in TSC22/2(MFI571.81)than in TSC21/2cells(MFI55.22) (Figure1A).Most of the CD9appeared to be concentrated at the plasma membrane,with a small amount located within the cells (Figure1B).There appeared to be more intracellular CD9in the null than in the heterozygous cells.Altogether,these data prompted us to isolate cells from bodyfluids based in part on the presence of this cell surface antigen.Detection of TSC2LOH in CD44v61/CD91Cellsfrom BALF and UrineCells from BALF,urine,and chylous effusion were incubated with anti-CD9and anti-CD44v6antibodies and separated by cell sorting.We found that the percentage of cells reactive with anti-CD44v6and anti-CD9antibodies differed considerably among bodyfluids and among patients,and ranged from0.51 to6.13%in BALF samples(n512),0.12to8.25%in urine samples(n555),and0.10to0.55%in chylous effusions(n5Cai,Pacheco-Rodriguez,Fan,et al.:Characterization of Disseminated TSC22/2Cells in LAM14115).Genomic DNA from these cells was isolated to determine TSC2LOH using microsatellites near the TSC2locus (i.e.,D16S291[z 331kb centromeric to TSC2],Kg8[z 100bp centromeric to TSC2],D16S3395[z 96kb telomeric to TSC2],D16S3024[z 443kb telomeric to TSC2],and D16S521[z 981kb telomeric to TSC2]).We specified that a cell population had TSC2LOH when at least one of the informative markers (heterozygosity of the alleles)had a Q LOH of less than 0.5(see M ETHODS ).Figures 2A and 2C show representative FACS plots of BALF and urine cell samples from a single patient,respectively,which were incubated with anti-CD44v6and anti-CD9antibodies.The CD44v61CD91cell population from BALF showed TSC2LOH at the Kg8microsatellite (Figure 2B,bottom panel ),as did a CD44v61CD91cell population from urine (Figure 2D,bottom panel ).Furthermore,we were able to determine that CD44v61CD91cells from chylous effusions had TSC2LOH (Table 1).These data suggest that LAM cells could be identified in BALF,urine,and chyle based on the expression of the tetraspanin CD9and the splice variant of the hyaluronic receptor,CD44v6.We had reported TSC2LOH in cells grown from LAM lung nodules and sorted with anti-CD44and anti-CD44v6antibodies (21),but LOH was not consistently seen in cells similarly separated from BALF (1out of 6)and urine (2out of 15)(see Figure E1in the online supplement).Populations reactive with anti-CD45/CD235a antibodies were not seen in BALF and urine cell samples (Figure E2),nor were those reactive with anti-CD44v6/CD44and anti-CD44v6/CD9antibodies seen in blood cell fractions (Figure E3).Thus,LAM cells in different locations appear to show differences in surface protein expression,which is consistent with their phenotypic heterogeneity in different M cells in lung nodules appear both spindle-shaped andepithelioid,whereas TSC22/2cells in renal AMLs may resemble adipocytes,vasculature,or smooth muscle cells.In our series of 55patients (Table E1),detection of TSC2LOH in blood fractions,urine,and BALF was markedly enhanced after cell sorting with the specified cell surface markers (Table 1and Figure 3).Despite the ability to show allelic imbalance and LOH in unsorted cells from urine and blood fractions,we failed to find TSC2LOH in cell pellets isolated from BALF (Figure 3and Table 1).Percentage of detection of TSC2LOH,however,was increased from 34%(18of 53patients)in unsorted blood samples separated using the OncoQuick density-gradient system to 90%(47of 52patients)after sorting with anti-CD45and anti-CD235a antibodies (P ,0.001).Cells from urine with TSC2LOH were more readily detected after sorting with anti-CD44v6and anti-CD9antibodies (from 13%[7of 53patients]to 69%[36of 52patients];P ,0.001).Furthermore,the success rate of detection of TSC2LOH was 70%(7of 10patients)in the CD44v61CD91cell population separated from BALF samples (P 50.003).To assess the reproducibility of detection of cells with TSC2LOH,blood and urine samples from 10patients with S-LAM at two visits,separated by 6to 18months,were analyzed andfoundFigure 1.CD9protein in TSC22/2fibroblastic cells grown from human tuberous sclerosis complex (TSC)-associated skin tumors.(A )Flow cytometric analysis showed significantly higher levels of CD9(clone M-L13,black histogram )as assessed by mean fluorescence intensity (MFI)in TSC22/2cells (MFI 571.81)than in TSC21/2cells (MFI 55.22).Gray histogram ,negative control.(B )Immunostaining analysis of TSC 21/2and TSC22/2skin cells with anti-CD9antibody.Reactivity was greater in TSC 22/2than in TSC21/2cells.Blue ,nuclear staining (DAPI).Bar,20m m.Experiments were replicated threetimes.Figure 2.Fluorescence-activated cell sorting (FACS)of cells in bron-choalveolar lavage fluid (BALF)and urine samples from a patient with lymphangioleiomyomatosis (LAM).Cells were reacted with anti–CD44v6-FITC and anti–CD9-PE antibodies and sorted cells were analyzed for TSC2loss of heterozygosity (LOH)of chromosome 16p13.3microsatellite marker Kg8.Four populations of BALF cells were separated (A ),and LOH was detected only in the CD44v61CD91population (B ,lowest histogram ).Two populations of urine cells were separated:CD44v62CD92and CD44v61CD91(C ).LOH was observed only in the CD44v61CD91population (D ,lowest histogram ).Arrows indicate positions of allelic loss compared with that seen in whole blood from the same patient.Numbers at the top of histograms indicate the number of DNA bases and the Y-axis indicates relative fluorescence units (RFU).1412AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 1822010to show reproducible detection of TSC2LOH using the same microsatellites in100%of blood samples and80%of urine samples(Table2and Figure E4).TSC2LOH Patterns in LAM Cells from Different Body Fluids Because the microsatellite markers on chromosome16p13.3near the TSC2gene cover a large region,we compared the patterns of TSC2LOH in samples from different sources in the same patients.Twenty-seven of47patients showed LOH at one informative microsatellite,and20of47patients at two informa-tive microsatellites,which involved different regions of chromo-some16(Table E1).We found that CD44v61CD91cells from BALF,urine,and chylous effusions and CD452CD235a2or CD452CD235a1cells from blood fractions showed,in general, identical TSC2LOH patterns,that is,loss or retention of the alleles at the same microsatellite loci(Table E1).Exceptions were observed,however,in eight cases of S-LAM and two of TSC-LAM,in which LAM cells from different bodyfluids did not show consistent LOH or ROH for each informative marker(Table E1, see S-LAM2,11,23,27,30,40,41,45,and TSC-LAM5,8).In addition,we observed that allelic loss in LAM cells from different bodyfluids consistently occurred at the same alleles for the same microsatellite markers(Figures2B and2D).We analyzed also the frequency of detection of LOH with different chromosome16p13.3microsatellite markers(Table3). The TSC2gene is closest to the Kg8microsatellite;other markers are more distant(D16S3395,D16S291,D16S3024 ,D16S521)and span a large region from approximately981kb telomeric through approximately331kb centromeric to the TSC2gene.Percentage of LOH detection by PCR using Kg8in informative patients approached97%in blood(n534),71%in urine(n535),and100%in BALF(n55),whereas percentage for other markers were significantly lower(P,0.05).Isolated LOH was observed both centromeric and telomeric to the TSC2 gene.FACS of Samples from Patients with LAM and Healthy Volunteers with Anti-CD44v6and Anti-CD9Antibodies Investigating whether identification of CD44v61CD91cells with TSC2LOH would distinguish patients with LAM(S-LAM or TSC-LAM)from healthy volunteers,we found that using similar cell fractionation and sorting,cells reactive with anti-CD44v6and anti-CD9antibodies were also seen in BALF and urine samples from healthy volunteers.We did not observe significant differences in reactivity to anti-CD44v6and anti-CD9antibodies among cells from S-LAM,TSC-LAM,and healthy volunteers.Table4presents data for patients with S-LAM(45blood and urine,10BALF,and5chylous effusions), patients with TSC-LAM(10blood and urine,and2BALF),and healthy volunteers(13blood,urine,and BALF).Of importance regarding the specificity of TSC2LOH,LOH was not detected in blood,urine,and BALF specimens from healthy volunteers (data not shown).We observed TSC2LOH in38of43(88%) blood specimens,28of42(67%)urine specimens,5of8(63%) BALF specimens,and2of4(50%)chylous effusions from informative patients with S-LAM.In patients with TSC-LAM, LOH was detected in9of9(100%)blood specimens,8of10 (80%)urine specimens,and2of2(100%)BALF specimens. The frequency of detection of TSC2LOH was greater in patients with TSC-LAM than in patients with S-LAM,but it was not statistically significant.The overall detection rate in patients with LAM was90%in blood,69%in urine,and70%in BALF.TSC2LOH was not found infive informative patients with S-LAM;two patients with S-LAM were noninformativeTABLE1.DETECTION OF TSC2-RELATED LOSS OF HETEROZYGOSITY IN DIFFERENT CELL POPULATIONS FROM DIFFERENT BODY FLUIDS OF PATIENTSWITH LYMPHANGIOLEIOMYOMATOSISType of LAM Fluid Cell Population Number ofInformativeSamples*%Sampleswith LOH†S-LAM Blood fraction Unsorted4335CD452/CD235a23985CD452/CD235a13686 Urine‡Unsorted4314CD44v62/CD92420CD44v61/CD914267 BALF Unsorted80CD44v62/CD9280CD44v61/CD9280CD44v62/CD9180CD44v61/CD91863 Chyle Unsorted40CD44v62/CD9240CD44v61/CD91450 TSC-LAM Blood fraction Unsorted1030CD452/CD235a2989CD452/CD235a19100 Urine‡Unsorted1010CD44v62/CD92100CD44v61/CD911080 BALF Unsorted20CD44v62/CD9220CD44v61/CD9220CD44v62/CD9120CD44v61/CD912100 Definition of abbreviations:BALF5bronchoalveolar lavagefluid;LAM5 pulmonary lymphangioleiomyomatosis;LOH5loss of heterozygosity;PCR5 polymerase chain reaction;S-LAM5sporadic LAM;TSC5tuberous sclerosis complex.*Samples were collected from45patients with S-LAM and10patients with TSC-LAM,but only samples that were heterozygous for the markers tested and that were amplified well by PCR were included.†Results of PCR assays are based on total offive microsatellite markers on chromosome16p13.3:D16S291,Kg8,D16S3395,D16S3024,and D16S521.‡Due to limited cell numbers in urine specimens,simultaneous sorting of four cell populations led to too few cells collected for each population to be analyzed for TSC2LOH.We randomly selectedfive cases that showed TSC2LOH in CD44v61CD91cells from urine to separate subsequently CD44v61CD92and CD44v62CD91cells,but did not identify TSC2LOH in thesepopulations.due to homozygosity of allfive tested markers.We further analyzed these patients using two single-nucleotide polymorphisms (SNPs)within the TSC2gene.Three patients with S-LAM (S-LAM15,35,39)were informative for one of two SNPs.LOH at the exon40polymorphism was detected in CD44v61CD91 cells from BALF of S-LAM39(data not shown),increasing the overall detection rate to80%in BALF samples.Because LAM is believed to result from mutations in the TSC1or TSC2gene,we looked to see if those lacking TSC2 LOH had TSC1LOH.We therefore assessed microsatellite markers at the TSC1locus,but none of these samples showed TSC1LOH(Table E2).In addition,patients with TSC2ROH in blood consistently showed ROH in urine and/or BALF. TSC2LOH and Clinical Phenotypes of Patients with LAM LAM is characterized by renal AMLs,lymphatic abnormalities, and pulmonary cystic lesions.We therefore assessed the associ-ation between the presence of AMLs and lymphatic involvement, which may represent the presence of more metastatic cells,and detection of LOH in cells from blood and urine.LOH was detected in86%(n528)of urine samples from patients with AMLs,but in only50%(n524)of urine samples from those without AMLs(P50.007),although the frequency of TSC2LOH was not significantly higher in blood cell fractions from patients with LAM with AMLs(n529)than in those without AMLs(n5 24;P50.08)(Figure4).These data suggest that LAM cells in blood might be shed into the urine in patients with LAM,or that necrosis in the AMLs might result in TSC22/2cells in the urine. There was no significant association between the presence of lymphangioleiomyomas,adenopathy,or lymphangioleiomyo-mas/adenopathy in patients with LAM with or without AMLs and detection of TSC2LOH in blood and urine cell fractions (Figure E5).DISCUSSIONWe have identified two cell surface proteins,CD44v6and CD9, that are useful for isolation of disseminated LAM cells from BALF,urine,and chylous M cells from patients with S-LAM exhibit mutations and LOH most frequently in the TSC2locus.For the majority of patients with S-LAM,LAM cells isolated from blood,urine,BALF,or chyle of the same patients show identical TSC2LOH patterns for specific microsatellites, although in some,LAM cells from different bodyfluids appeared to differ in the extent of TSC2LOH regions,consistent with genetic heterogeneity.TABLE2.TSC2-RELATED LOSS OF HETEROZYGOSITY IN BLOOD AND URINE FROM PATIENTS ON TWO VISITSCase no.Visit Interval(mo)FluidAllelic Status of TSC2-Related MicrosatellitesD16S291Kg8D16S3395D16S3024D16S521S-LAM11Blood NA ROH LOH NI LOH17Urine NA ROH ROH ROH 2Blood NA ROH LOH LOHUrine NA ROH ROH ROH S-LAM21Blood NA NI LOH NA NI18Urine NA ROH NA2Blood NA LOH NAUrine NA ROH LOHS-LAM81Blood NI LOH LOH NI NI12Urine LOH NA2Blood LOH NAUrine LOH ROHS-LAM91Blood NI NA NI NA LOH18Urine ROH ROH LOH 2Blood NA NA LOHUrine ROH ROH LOH S-LAM271Blood NI LOH LOH NI NI6Urine LOH ROH2Blood LOH LOHUrine ROH ROHS-LAM281Blood NI LOH NA NI NI12Urine LOH ROH2Blood LOH NAUrine LOH ROHS-LAM301Blood NI LOH LOH NA NI12Urine LOH ROH NA2Blood LOH LOH NAUrine LOH ROH NAS-LAM311Blood NI NI LOH LOH LOH12Urine LOH LOH LOH 2Blood LOH NA NAUrine LOH NA LOH S-LAM401Blood NI LOH LOH NI NA14Urine ROH ROH LOH 2Blood LOH NA NAUrine ROH NA NAS-LAM431Blood NI LOH NI NI NI13Urine LOH2Blood LOHUrine ROHDefinition of abbreviations:LOH5loss of heterozygosity;NA5not amplified;NI5noninformative:homozygosity of the markers tested;ROH5retention of heterozygosity;S-LAM5sporadic lymphangioleiomyomatosis.1414AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL1822010In general,thesefindings support the hypothesis that multi-system manifestations of LAM appear to result from a meta-static process(31).Previously infive patients with S-LAM, identical TSC2mutations or LOH were identified in pulmonary and renal LAM lesions(13–15).LAM cells from the recipient were identified in a transplanted donor lung(17,18).As LAM cells were also detected in blood(19)or chylousfluids(19,20), it was hypothesized that LAM cells could migrate or metasta-size via blood and/or lymphatic circulations(19,20).The LAM cells found in BALF could result from release from the LAM lung nodules or perhaps shedding from the lymphatic circula-tion within the nodules.Patients with LAM may experience M cells were identified in chyle and BALF may contain components of chyle.Identification of proteins on the surface of circulating or disseminated cells has been of interest in human cancers for use potentially as therapeutic targets(32–34).Gene expression micro-array analysis revealed that TSC22/2cells grown from TSC-associated skin tumors contained highly increased mRNA levels of the tetraspanin CD9(25).We demonstrated here usingflow cytometric analysis and immunostaining that CD9protein was abundant on TSC22/2skin tumor cells(Figure1).A correlation between greater CD9content and potential for metastasis is evident in some tumor types(e.g.,bone,cervix,head and neck, stomach)(35),although CD9is considered to suppress metastasis by decreasing cell motility(35–38).Tetraspanins are present widely among mammals and play important roles in cell morphology, motility,invasion,adhesion,and signaling(39–42).Tetraspanins form complexes with other tetraspanins and a variety of trans-membrane proteins at tetraspanin-enriched membrane microdo-mains(42,43).The diverse actions of CD9are probably due to its association with other molecules in the tetraspanin-enriched membrane microdomains.In our studies,high levels of CD9 protein correlated with cells having TSC2LOH(Figure1).Our group had reported earlier an association between the presence of CD44v6protein and TSC2LOH in LAM cells grownTABLE3.FREQUENCY OF DETECTION OF TSC2-RELATED LOSS OF HETEROZYGOSITY BY POLYMERASE CHAIN REACTION AMPLIFICATION OF DIFFERENT MICROSATELLITES ON CHROMOSOME16P13.3MarkerPositions of TSC2andMarkers on Chromosome16p13.3*Distanceto TSC2FluidNumberof Cases†Number ofInformative Cases‡%with LOH inInformative CasesD16S291TABLE4.DETECTION OF TSC2-RELATED LOSS OF HETEROGEITY IN CELL SAMPLES SORTED FROM BODY FLUIDS FROM PATIENTS WITH SPORADIC-LYMPHANGIOLEIOMYOMATOSIS AND TUBEROUS SCLEROSIS COMPLEX–LYMPHANGIOLEIOMYOMATOSIS,AND FROM HEALTHY VOLUNTEERSGroup Fluid*Number of Cases Noninformative†Not AmplifiedInformative‡%Patients with LOH ROH LOH All Cases Informative CasesS-LAM Blood45205388488 Urine452114286267BALF1020355063Chyle501224050TSC-LAM Blood10010990100 Urine1000288080BALF20002100100 Healthy volunteers Blood130211000 Urine130112000BALF130013000Definition of abbreviations:BALF5bronchoalveolar lavagefluid;LAM5pulmonary lymphangioleiomyomatosis;LOH5loss of heterozygosity;PCR5polymerase chain reaction;ROH5retention of heterozygosity;S-LAM5sporadic LAM;TSC5tuberous sclerosis complex.*Cells separated from blood by OncoQuick density-gradient centrifugation were reacted with anti–CD45-FITC and anti–CD235a-PE antibodies and cells from urine, BALF,and chylous effusions with anti–CD44v6-FITC and anti–CD9-PE antibodies.†Noninformative:homozygosity of the markers tested.‡Results of PCR assays are based on a total offive microsatellite markers on chromosome16p13.3:D16S291,Kg8,D16S3395,D16S3024,and D16S521.Cai,Pacheco-Rodriguez,Fan,et al.:Characterization of Disseminated TSC22/2Cells in LAM1415。
