㊀㊀基金项目:国家自然科学基金资助项目(81500893)作者单位:222002㊀徐州医科大学附属连云港医院口腔科通讯作者:秦晗,主任医师,硕士生导师,电子信箱:qinhan2005@RANKL ㊁OPG ㊁NF -κBp65蛋白表达在牙龈癌中临床意义秦㊀晗㊀龚永庆㊀徐宏志摘㊀要㊀目的㊀观察牙龈癌中RANKL㊁OPG㊁NF -κBp65蛋白表达和RANKL /OPG 比值变化,初步探讨骨质破坏相关蛋白所代表的生物学行为和临床意义㊂方法㊀收集2018年7月~2019年12月笔者医院牙龈癌临床标本,采用免疫组织化学方法检测RANKL㊁OPG㊁NF -κBp65蛋白表达并分析RANKL /OPG 比值变化㊂通过Western blot 法比较体外培养的牙龈癌和牙龈上皮细胞中3种蛋白表达及RANKL /OPG 比值差异㊂结果㊀牙龈癌临床标本中,RANKL 和OPG 蛋白在细胞质中强阳性表达,NF -κBp65在细胞核中强阳性表达,对照组弱阳性表达,实验组与对照组比较,差异有统计学意义(P <0.05),RANKL /OPG 比值较对照组升高,差异有统计学意义(P <0.05)㊂Western blot 法结果显示,与牙龈上皮细胞比较,牙龈癌细胞中RANKL㊁OPG㊁NF -κBp65蛋白过表达,RANKL /OPG 比值升高,差异有统计学意义(P <0.05)㊂结论㊀RANKL㊁OPG 蛋白表达及RANKL /OPG 比值升高后可能通过激活NF -κBp65引起牙龈上皮细胞异常分化为牙龈癌细胞,为牙龈癌发病机制探讨及临床治疗靶点选择提供新的实验依据㊂关键词㊀牙龈癌㊀骨质破坏相关蛋白㊀临床意义中图分类号㊀R34㊀㊀㊀㊀文献标识码㊀A㊀㊀㊀㊀DOI ㊀10.11969/j.issn.1673-548X.2021.02.012Expression of RANKL ,OPG ,NF -κBp65Protein in Gingival Carcinoma and Its Clinical Significance.㊀Qin Han ,Gong Yongqing ,XuHongzhi.Department of Stomatology ,The Lianyungang Affiliated Hospital of Xuzhou Medical University ,Jiangsu 222002,ChinaAbstract ㊀Objective ㊀To detect the expression ofRANKL,OPG,NF -κBp65protein in gingival carcinoma,and to observe the change of RANKL /OPG ratio,then to investigate the biological behavior and clinical significance of bone destruction associated protein.Methods ㊀The expression of RANKL,OPG and NF -κBp65protein was detected by immunohistochemical method in the clinical speci-mens of gingival carcinoma from July 2018to December 2019,and RANKL /OPG ratio was analyzed.Western blot was used to compare the expression of three proteins in human gingival cancer cells and gingival epithelial cells,and to analyze the RANKL /OPG ratio.ResultsIn the clinical specimens of gingival carcinoma,RANKL and OPG were strongly positive in the cytoplasm,NF -κBp65was strongly posi-tive in the nuclei.The control group was weakly positive by immunohistochemical pared with the control group,the experi-mental group had statistical significance(P <0.05).The ratio of RANKL /OPG was significantly higher than that of control group (P <0.05).Western blot analysis showed that RANKL,OPG,NF -κBp65protein overexpression and RANKL /OPG ratio was significantly higher in gingival cancer cells than in gingival epithelial cells,the results were statistically significant (P <0.05).Conclusion ㊀The in-creased expression of RANKL,OPG and RANKL /OPG ratio may induce abnormal differentiation of gingival epithelial cells into gingival cancer cells by activating NF -κBp65,which provides a new experimental basis for the study of the pathogenesis of gingival cancer and theselection of clinical therapeutic targets.Key words ㊀Gingival carcinoma;Bone destruction associated protein;Clinical significance㊀㊀牙龈癌发生率高且易早期侵犯颌骨引起骨质破坏,目前主要以手术切除为主配合放化疗,治疗效果欠佳[1]㊂近年发展起来的靶向治疗因在多种肿瘤中疗效显著已使其成为新的有效治疗手段[2]㊂研究牙龈癌发生过程中分子机制,寻找有效治疗靶点,对于牙龈癌的彻底治疗具有重要意义㊂核因子-κB 受体活化因子配体(RANKL)属于肿瘤坏死因子超家族成员,通过与核因子-κB 受体活化因子(RANK)结合,促进破骨细胞分化成熟,发挥骨吸收作用㊂骨保护蛋白(OPG )是诱饵受体,与RANKL 结合后可阻断RANKL /RANK 结合[3,4]㊂局部微环境中RANKL 和OPG 表达的相对水平即RANKL /OPG 比值是决定破骨细胞形成和活性的关键,若比值升高,则破骨细胞形成活跃,反之则破骨细胞形成受抑[5]㊂㊃84㊃近年来RANKL与肿瘤之间关系引发关注,研究表明,大多数肿瘤细胞中RANKL处于持续性激活状态,抑制RANKL可阻止肿瘤生长和肿瘤转移引起的骨质破坏,因此认为RANKL与肿瘤的发生㊁分化㊁侵袭和转移密切相关,可作为肿瘤诊断标志物和有效治疗靶点[6]㊂NF-κB是重要转录因子,主要以p65/ p50二聚体形式与抑制因子(I-κB)绑定并长期处于细胞质中㊂当细胞膜受体相应配体刺激后,I-κB激酶通过泛素-蛋白酶体通路介导I-κB复杂磷酸化,使p65/p50二聚体被释放进入细胞核,从而启动对目标基因的转录[7,8]㊂NF-κBp65参与NF-κB通路激活的经典和非经典通路中,被视为NF-κB通路激活的重要证据㊂本研究将RANKL㊁OPG㊁NF-κBp65蛋白和RANKL/OPG比值变化作为研究对象,以期揭示牙龈癌的发生可能与RANKL过表达后激活NF-κBp65,进而通过NF-κB通路正反馈机制引起牙龈上皮细胞异常分化相关㊂材料与方法1.主要材料:牙龈癌临床标本(徐州医科大学附属连云港医院)㊁牙龈癌和牙龈上皮细胞系(中国科学院上海细胞库)㊁胎牛血清(上海威正翔禹生物科技有限公司)㊁免疫组化试剂盒(北京中杉金桥生物科技有限公司)㊁BCA蛋白检测试剂盒(上海碧云天生物科技有限公司)㊁蛋白质预染标志物(上海赛默飞世尔有限公司)㊁ECL-PLUS试剂盒(上海赛默飞世尔有限公司)㊁SDS-PAGE蛋白电泳仪(上海天能科技有限公司)㊁蛋白转膜仪(上海天能科技有限公司)㊁离心机(上海赛默飞世尔有限公司)㊁倒置显微镜(上海蔡康光学仪器有限公司)㊁生物安全柜(上海振样创科技有限公司)㊁CO2培养箱(日本三洋贸易株式会社)㊂2.免疫组化检测临床标本:收集2018年7月~ 2019年12月笔者医院诊治的牙龈癌标本,制备组织切片,脱蜡至水,染色前60ħ放置1h㊂按试剂盒说明进行免疫组化染色,分别加入RANKL[小鼠,美国Proteintech公司,66610-1-Ig,1ʒ500,(35~ 38)kDa]㊁OPG(兔,英国Abcam公司,ab9986,1ʒ500, 56kDa)㊁NF-κBp65(兔,美国CST公司,#8242, 1ʒ100,65kDa)多克隆抗体,4ħ冰箱过夜,标本彻底清洗后加入稀释好的兔抗或鼠抗IgG抗体(兔,美国CST公司,#7074,1ʒ2000,小鼠,#7076,1ʒ2000)孵育30min㊂苏木精复染,梯度乙醇脱水,二甲苯透明,中性树胶封片,显微镜下观察RANKL㊁OPG㊁NF-κBp65蛋白表达并分析RANKL/OPG比值㊂3.牙龈癌细胞和牙龈上皮细胞培养:从液氮罐中取出购自中国科学院上海细胞库的人牙龈癌细胞和牙龈上皮细胞冻存管,完全解冻后,1300r/min,离心3min㊂而后吸去上清,加入MEM培养基重悬细胞,晃匀后置于37ħ㊁5%CO2培养箱进行细胞培养㊂24h后更换培养液,待细胞汇合度达80%左右传代㊂继续培养至细胞对数生长期时,用胰蛋白酶消化制成(3~5)ˑ104个/毫升单细胞悬液,以2ˑ104/cm2的密度接种到相应培养板,继续培养24h㊂4.Western blot法检测RANKL㊁OPG㊁NF-κBp65蛋白表达:收集牙龈癌和牙龈上皮细胞,PBS洗涤, 2ˑLysis Buffer裂解,4ħ㊁12000r/min㊁离心15min,而后取上清,BCA法测定蛋白浓度㊂根据目的蛋白大小配制不同浓度胶,采用十二烷基硫酸钠聚丙烯酰胺凝胶法进行电泳㊂电泳结束后,使用转移电泳装置, 4ħ㊁300mA恒流电转150min,将蛋白转移到PVDF 膜上㊂分别将稀释好的RANKL(1ʒ2000)㊁OPG(1ʒ2000)㊁NF-κBp65(1ʒ500)多克隆抗体(厂家批号同免疫组化)与封闭好的PVDF膜孵育过夜㊂彻底清洗后再将稀释好的兔抗或鼠抗IgG(1ʒ2000)抗体(厂家批号同免疫组化)与PVDF膜孵育1.5h㊂再次清洗PVDF膜后X线显影㊁定影㊁晾干㊁结果分析㊂内参为GAPDH(小鼠,上海SANTA CRUZ生物技术有限公司,sc-32233,1ʒ2000,36kDa)㊂5.统计学方法:应用SPSS19.0统计学软件对实验组和对照组数据进行t检验,以P<0.05为差异有统计学意义㊂结㊀㊀果1.免疫组化结果:分别在实验组牙龈癌细胞核和细胞质中见到棕色沉淀分布,RANKL㊁OPG在细胞质中强阳性表达,NF-κBp65在细胞核中强阳性表达㊂对照组免疫组化染色弱阳性(图1)㊂与对照组比较, RANKL㊁OPG㊁NF-κBp65蛋白表达及RANKL/OPG 比值升高,差异有统计学意义(P<0.05,图2㊁图3)㊂2.Western blot法检测结果:与对照组比较,实验组RANKL㊁OPG㊁NF-κBp65蛋白表达均升高(图4)㊂与对照组比较,RANKL㊁OPG㊁NF-κBp65蛋白表达及RANKL/OPG比值升高,差异有统计学意义(P<0.05,图5㊁图6)㊂讨㊀㊀论RANKL是肿瘤坏死因子超家族成员,存在膜结合和分泌型两种形式,可与破骨前体细胞RANK相结合,诱导破骨细胞成熟㊂以往研究认为,RANKL参㊃94㊃图1㊀免疫组化法检测牙龈癌蛋白表达(ˑ200)A.实验组RANKL 染色;B.对照组RANKL 染色;C.实验组OPG 染色;D.对照组OPG 染色;E.实验组NF -κBp65染色;F.对照组NF -κBp65染色图2㊀免疫组化检测牙龈癌蛋白表达统计学分析㊀图3㊀免疫组化法检测RANKL /OPG 蛋白比值统计学分析㊀与骨代谢㊁免疫㊁心血管等多种生理病理过程,近年来RANKL 与肿瘤间关系越发受到重视[9,10]㊂大量研究表明,大多数肿瘤细胞中RANKL处于持续性激活状图4㊀Western blot 法检测牙龈癌细胞RANKL ㊁OPG ㊁NF -κBp65蛋白表达结果A.RANKL;B.OPG;C.NF -κBp65;D.内参;1㊁3㊁5为对照组,2㊁4㊁6为实验组㊀图5㊀Western blot 法检测牙龈癌细胞RANKL ㊁OPG ㊁NF -κBp65蛋白表达统计学分析㊀图6㊀Western blot 法检测RANKL /OPG蛋白比值统计学分析㊀态,因此认为RANKL 与肿瘤发生㊁发展密切相关,这将为相关疾病治疗提供新的思路和方法㊂通过对多发性骨髓瘤研究发现,在骨髓瘤细胞和骨髓间质细胞内均可见RANKL 表达升高[11]㊂乳腺癌研究中提示RANKL 升高可能与乳腺癌细胞表达或诱导多种促进骨代谢因子,致使骨髓基质细胞大量产生RANKL 有㊃05㊃关[12]㊂通过临床标本和体外培养的牙龈癌细胞研究显示,牙龈癌细胞中RANKL表达较对照组明显升高,提示同大多数肿瘤一样,牙龈癌中RANKL也处于激活状态,这为本研究的最初设想即将RANKL作为牙龈癌的治疗靶点提供有效的实验依据㊂OPG作为诱饵受体,主要功能是抑制破骨细胞分化㊁成熟,并诱导其凋亡㊂作用机制为直接抑制RANK/RANKL复合体活性,或者通过与RANK竞争性结合RANKL,间接抑制骨吸收以及OPG独立于RANKL直接抑制破骨细胞活性,抑制骨吸收[13,14]㊂人体内骨代谢平衡或失调更多取决于RANKL/OPG 比值,而不是RANKL或OPG绝对含量值,比值降低,破骨细胞分化和活性降低,反之则升高㊂介于RANKL和OPG相互拮抗作用,因此观察RANKL/ OPG比值变化较单独观察RANKL和OPG变化更有意义[15]㊂本研究对临床标本和体外培养牙龈癌细胞中OPG蛋白检测发现,与对照组比较,实验组OPG 蛋白表达升高,但牙龈癌中RANKL/OPG比值仍高于对照组㊂此结果提示当RANKL和OPG表达量均升高而骨吸收没有被相应抑制时,可能是因为牙龈癌细胞中RANKL表达远远高于OPG的表达,即RANKL/ OPG比值增加的缘故㊂NF-κB调节免疫球蛋白κ轻链在B淋巴细胞中的表达,若失调易导致各种恶性肿瘤发生,因此,很多研究着力于探索实体瘤中NF-κB表达的临床意义,以提示疾病预后[16]㊂NF-κB主要以p65/p50二聚体形式存在于细胞内,未激活时,NF-κB与I-κB 结合,存在于细胞质中,若受到细胞膜外信号刺激, I-κB激酶复合体将I-κB磷酸化,使NF-κB暴露核定位点㊂游离的NF-κB迅速移位到细胞核,诱导相关基因转录[17]㊂NF-κB信号通路可以被RANKL 等炎性介质激活,其活化后也可以作为外界刺激因子进一步活化NF-κB,形成NF-κB自身活化的正反馈机制[18]㊂p65参与NF-κB激活的经典和非经典通路,被视为NF-κB通路激活的重要证据[19]㊂本研究选择NF-κBp65作为牙龈癌检测指标,旨在观察NF-κBp65激活是否与局部微环境中RANKL/ OPG比值增加有关,以及NF-κB信号通路是否是牙龈癌发生的重要信号通路,进而分析靶向RANKL治疗牙龈癌时NF-κBp65表达变化与临床效果和生存的关系㊂笔者临床标本和体外培养细胞的研究结果同时提示,与正常对照组比较,牙龈癌中NF-κBp65存在过表达现象,提示局部微环境中RANKL/OPG比值增加可能导致NF-κB信号通路激活进而引起牙龈上皮细胞异常分化㊂近年来虽然牙龈癌的基础和临床诊疗方法的研究取得很大进展,但治疗效果未见显著提高[20]㊂靶向治疗是针对细胞受体㊁关键基因㊁调控分子等生物靶点进行干预,达到阻止肿瘤发生㊁发展并促进肿瘤细胞凋亡的特异性治疗,因其发展迅速㊁疗效较佳,引起众多学者关注[21]㊂因此,明确牙龈癌发生过程中调控牙龈上皮细胞异常分化的分子信号机制对于牙龈癌有效治疗靶点的选择至关重要㊂本研究通过观察RANKL㊁OPG㊁NF-κBp65蛋白表达及RANKL/ OPG比值变化,初步探讨牙龈癌细胞分化的可能调控机制,从而为寻找牙龈癌有效治疗靶点提供帮助㊂参考文献1㊀Sato K,Hayashi Y,Watanabe K,et al.Concurrent chemoradiothera-py with intravenous cisplatin and docetaxel for advanced oral cancer [J].Nagoya J Med Sci,2019,81(3):407-4142㊀Qiao M,Zhao C,Liu Q,et al.Impact of ALK variants on brain me-tastasis and treatment response in advanced NSCLC patients with on-cogenic ALK fusion[J].Transl Lung Cancer Res,2020,9(4): 1452-14633㊀Ibrahim T,Ricci M,Scarpi E,et al.RANKL:a promising circulat-ing marker for bone metastasis response[J].Oncol Lett,2016,12 (4):2970-29754㊀de Castro LF,Burke AB,Wang HD,et al.Activation of 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