PCNA反义寡核苷酸抑制食管鳞癌细胞株T.Tn体外增殖活性的实验研究  

In Vitro Experiment of Inhibiting Cell Proliferation on Human Esophageal Squamous Cell Cancer T.T n Cell Line by Proliferating Cell Nuclear Antigen Antisense Olig odeoxynucleotide

作　　者：杨林[1] 叶世铎[1] 冯伦高[1] 王家顺[1] 翟伟[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院胸外科,湖北武汉430022

出　　处：《癌症》2002年第3期276-280,共5页Chinese Journal of Cancer

摘　　要：背景与目的:增殖细胞核抗原(proliferationcellnuclearantigen,PCNA)在食管鳞癌患者中的高表达提示患者的预后不良。本研究拟通过基因反义封闭技术体外抑制PCNA的表达,以达到抑制肿瘤细胞恶性增殖的目的。方法:以食管鳞癌T.Tn细胞株为研究对象,采用硫代磷酸修饰的PCNA反义寡核苷酸(antisenseoligodeoxynucleotide,ASODN),通过脂质体导入T.Tn细胞后,观察PCNAASODN对T.Tn细胞癌基因表达及体外增殖活性的影响。结果:相差显微镜下观察到T.Tn细胞转染ASODN后细胞体积变小,皱缩变形的细胞较对照组明显减少。MTT法测细胞增殖活性见不同浓度ASODN均能抑制T.Tn细胞的增殖,抑制作用在24h最强,0.2μmol/LASODN对细胞的抑制率为63.3%,时间延长抑制作用逐渐减弱。3H-TdR掺入实验表明ASODN能有效地抑制T.Tn细胞的DNA合成。免疫组化SABC法染色提示转染ASODN后T.Tn细胞mRNA的翻译受到抑制,PCNA蛋白表达水平明显降低。FCM检测发现G0-G1期细胞含量由对照组的53.65%增至72.34%,而G2-M和S期细胞则分别从15.51%、30.84%降到9.87%、17.79%。表明PCNAASODN转染导致PCNA促进细胞增殖的功能降低,抑制处于G0期的细胞进入S期。结论:使用人工合成的PCNAASODN可以特异性地抑制食管鳞癌T.Tn细胞株PCNA蛋白的表达,从而调控细胞周期。Background &Objectives:The high expression of proliferatio n cell nuclear antigen were found in t he patients with esophageal carcinoma in poor prognosis.The aim of this stu dy was to investigate the effect in vitro of antisense oligodeoxynucleotide(ASODN)of PCNA on its gene expression and cell proliferation in human esophageal squamous cell cancer T.Tn cell line.Methods :Phosphorothioate PCNA ASODN and sen se oligodeoxynucleotide(SODN)were synthesized and transfected to T.Tn cells mediated by dosper liposo mal.Their effects on cell prolifera tion were examined by MTT method,3 H-TdR method,immunohistochemical study,and flow cytometer(FCM).Results:PCNA ASODN could significantly inhi bit the growth of T.Tn cell lines.The SODN and control lipsome showed no su ch effect.The inhibition peaked at 24h our after transfection,the inhibition rate reached 63.3%at 0.2μmol /L by MTT method,the inhibition effect was dose-dependent.ASODN could downregu late the expression levels of PCNA pr otein.And PCNA ASODN transfection could in hibit the transition period of G0 /G1 phase to S phase.Conclusions :The data suggested that ASODN could specific inhibit the expression of PCNA prote in and regulate cell cycle and cell pr oliferation in T.Tn cells and antisense therapy c ould be an useful method to improve prognosis in ESCC.

关 键 词：食管肿瘤 鳞状细胞癌 增殖细胞核抗原 反义寡核苷酸 PCNA T.Tn 体外增殖 

分 类 号：R735.1[医药卫生—肿瘤]