乙型肝炎病毒X蛋白对HepG2细胞羧末端结合蛋白反应蛋白表达的影响  

The effect of hepatitis B virus X protein on the expression of CtlP in HepG2 Cells

作　　者：刘庆[1] 王梦漪[2] 何星星[1] 陈曼[1] 宋起龙[1] 蒋翔[1] 谢琼慧[1] 林菊生[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院肝病研究所,武汉430030 [2]华中科技大学同济医学院附属同济医院肿瘤科,武汉430030

出　　处：《中华肝脏病杂志》2011年第8期577-581,共5页Chinese Journal of Hepatology

基　　金：国家自然科学基金面上项目（30872237）

摘　　要：目的研究乙型肝炎病毒X蛋白（HBx）对博莱霉素诱导HepG2细胞DNA双链断裂（DSB）损伤修复关键因子羧末端结合蛋白反应蛋白（CtIP）的影响。方法建立稳定表达HBx的HepG2肝癌细胞株（HepG2-HBx）及空质粒对照细胞株（HepG2-vec）。用博莱霉素诱导细胞DSB损伤后，用流式细胞仪检测细胞周期和细胞凋亡情况，用实时定量PCR及Western blot检测CtIP的mRNA与蛋白表达水平，并用激光共聚焦显微镜观察CtIP蛋白在细胞内的定位情况。多组数据采用单因素方差分析和SNK—q检验；两组数据间比较用t检验。结果经检测转染HBx基因的HepG2细胞（HepG2-HBx）能稳定表达HBx。博莱霉素处理后，HepG2-HBx细胞与HepG2-vec细胞的凋亡比例分别为16．90％±0．89％和15．30％±0．86％，差异无统计学意义（q=2．074，P〉0．05），但死亡细胞比例分别为8．71％±0．74％和4．90％±0．46％，差异有统计学意义（q=7．126，P〈0．01）；同时两种细胞株都出现了细胞周期G2／M期阻滞，差异有统计学意义（F=11．401，P〈0．05）。HBx使CtIP蛋白表达水平和mRNA表达水平均下调，HeDG2-HBx细胞与HepG2-vec细胞CtIP蛋白的相对表达量分别为0．66±0．04、0．73±0．05，差异有统计学意义（t=2．314，P〈0．05）;CtIP mRNA相对表达量分别为1．00±0．06、1．23±0．08，差异有统计学意义（t=2．732，P〈0．05）。同时观察到CtIP蛋白主要在细胞胞核内表达。结论HBx能干扰肿瘤抑制蛋白CtIP的表达，可能影响细胞DSB损伤的修复。Objective To investigate the effect of hepatitis B virus X protein（HBx） on CtBP-interacting protein（CtIP） which is an important repair factor of DNA double strand break damage in HepG2 cells induced by bleomycin. Methods A HBx stably expressing HepG2 cell line and a control HepG2 cell line with empty vector transfected were established. After the double strand break （DSB） damage occurred, the mRNA and protein levels of CtIP were detected by Real-time PCR and Western blot assay respectively, cell cycle profiles and apoptotic cell death were determined by a flow cytometry, and the position of CtIP in ceils was observed by confocal laser scanning microscopy. Results It showed that HepG2 cells transfected with hepatitis B virus X gene could stably express HBx protein. After being induced by bleomycin, the percentage of apoptotic cell was 16.90% ± 0.89% in HBx stably expressing HepG2 cell line and 15.30% ±0.86% in control cell line, respectively（ q = 2.074, P 〉 0.05）. While the percentage of death cell was 8.71% ±0.74% in HBx stably expressing HepG2 cell line and 4.90% ±0.46% in control cell line, respectively（ q = 7.126, P 〈 0.01）. The two cell lines manifested the increase of G/M arrest and significant difference existed between the two cell lines. HBx down regulated the expression levels of CtIP and its mRNA. The CtIP level was 0.66 ±0.04 in HepG2-HBx cell and 0.73 ±0.05 in HepG2-vec cell, respectively（t= 2.314,P 〈 0.05）. The relative mRNA level was 1.00 ± 0.06 in HepG2-HBx cell and 1.23 ± 0.08 in HepG2-vec cell, respectively（ t = 2. 732, P 〈 0.05）. We also found that CtIP was concentrated in the cell nucleus. Conclusion The research suggests that HBx may affect DNA-repair pathways by disrupting the expression of CtIP.

关 键 词：癌 肝细胞 乙型肝炎病毒X蛋白 DNA双链断裂 修复 

分 类 号：R51[医药卫生—内科学] R3[医药卫生—临床医学]