乙肝病毒X基因对人肝癌细胞株HepG2凋亡的影响

目的 探讨乙肝病毒X基因(hepatitis B virus,HBX)对人肝癌细胞株HepG2凋亡的影响.方法 采用脂质体转染法将HBX真核表达载体pcDNA3.1-X转人人肝癌细胞HepG2中,建立表达HBX的HepG2/HBX细胞模型;以转染空载体pcDNA3.1的HepG2/pcDNA3.1细胞为对照,于转染后24、48、72、96 h收集细胞标本,行流式细胞术分析肝细胞凋亡率变化情况,RT-PCR、Western blot分别检测肝癌细胞内HBX和凋亡相关基因Fas、FasL表达以及JNK、c-Jun蛋白磷酸化水平情况.结果 转染24 h后HepG2/HBX细胞中即检测出HBX表达,且其表达量随时间延长而逐渐增高(P＜0.05);对照组细胞中各时间点均无HBX的表达.转染后各时间点,HepG2/HBX细胞与对照组细胞相比,细胞凋亡率均升高(P＜0.05),Fas、FasL表达量和JNK、c-Jan蛋白磷酸化水平亦均增高(P＜0.05);且HBX mRNA表达量与细胞凋亡率、Fas和FasL表达量以及JNK、c-Jun的磷酸化水平均呈正相关(P＜0.05).结论 HBx可通过上调JNK、c-Jun蛋白磷酸化水平而促进FasL蛋白的表达,从而诱导HepG2细胞凋亡.

Effect of Hepatitis B Virus X Gene on the Apoptosis of HepG2 Cells

Objective To investigate the effect of hepatitis B virus X gene on the apoptosis in X genetransfected HepG2 cells.Methods HBX gene eukaryon expression vector pcDNA3.1-X was transfected into HepG2 cells by lipid-mediated transfection to establish HepG2/HBX cell model for HBX expression.HepG2 transfected with pcDNA3.1 was used as controls.At 24 h,48 h,72 h and 96 h after transfection,cell apoptotic rates were detected by flow cytometry.RT-PCR and Western blot were applied to evaluate the expression levels of HBX,Fas,FasL,and the levels of p-JNK and p-c-Jun protein.Results HBX mRNA was detected in HepG2/HBX cells 24 h after transfection,and the expression of HBX mRNA level was gradually up-regulated after transfection(P＜0.05);Whereas no expression of HBX gene in the control cells.At 24 h,48 h,72 h and 96 h after transfection,all of the apoptotic rate in HepG2/HBX group were much higher than those in the controls(P＜0.05),The expression levels of Fas and FasL protein as well as the levels of p-JNK and p-c-Jun protein were significantly higher than those in the controls(P＜0.05).A positive correlationship was observed between the expression of HBX mRNA level and the hepatocyte apoptotic rate(P＜0.05),the same correlation of HBX mRNA level with the levels of Fas,FasL,p-JNK and p-c-Jun were also observed.(P＜0.05).Conclusion HBX can induce hepatocyte apoptosis by up-regulating the levels of p-JNK and p-c-Jun to promote the expression of FasL.