Objective: To investigate the influence of hepatitis B virus X gene (HBx) on apoptosis of hepatic cells mediated by Fas in HePG2 cells. Methods: HBx eukaryotic vector pcDNA3.1(+)-X was transfected into HEPG2 cells with lipofectamine, and the null vector pcDNA3.1(+) and untransfected HEPG2 were used as normal controls. The cells were collected 72 h after transfection, and the expression of HBx mRNA and protein was determined using RT-PCR and Western blot, respectively. The mRNA expression of apoptosis-related genes Bcl-2 and Bax mRNA was also determined using RT-PCR. Cytotoxicity and apoptosis were evaluated using CCK-8 and flow cytometry, respectively, after HepG2-HBx and HepG2-3.1 cells were treated with stimulatory monoclonal antibody anti-Fas CH11. The t test was used for pairwise comparison. Results: The cell line HepG2-HBx was successfully established, as confirmed by RT-PCR and Western blot, and RT-PCR results showed that HepG2-HBx cells had significantly higher expression of Bcl-2 mRNA than HepG2-3.1 and HepG2 cells (P < 0.05), but had significantly lower expression of Bax mRNA than HepG2-3.1 and HepG2 cells (P < 0.05); CCK-8 and flow cytometry showed that anti-Fas CH11 had a lower cytotoxicity to HepG2-HBx cells and allowed for a lower apoptosis rate of HepG2-HBx cells compared with HepG2-3.1 and HepG2 cells. Conclusions: HBx can inhibit apoptosis of hepatic cells mediated by the Fas pathway.
目的： 研究乙型肝炎病毒X基因（HBx）对HepG2细胞中Fas介导的肝细胞凋亡的影响。 方法： 用脂质体转染法将HBx真核表达载体pcDNA3.1(+) -X瞬时转入HepG2细胞，以转染空质粒pcDNA3.1(+)的细胞及未转染的HepG2细胞为对照。转染后72 h收集细胞，通过RT-PCR及Western blot法检测HBx mRNA和蛋白质的表达，并通过RT-PCR检测凋亡相关基因Bcl-2、Bax mRNA的表达量变化。采用激动型Fas单克隆抗体抗-Fas CH11分别作用于HepG2-HBx细胞和HepG2-3.1细胞后，通过CCK-8和流式细胞术检测细胞毒性和凋亡。两样本比较用t检验。 结果： RT-PCR及Western blot证实成功构建细胞株。HepG2-HBx且RT-PCR结果显示HepG2-HBx细胞中Bcl-2 mRNA相对表达量显著高于HepG2-3.1及HepG2（P < 0.05），而Bax mRNA相对表达量显著低于HepG2-3.1及HepG2 （P < 0.05）；CCK-8和流式细胞术检测结果显示抗-FasCH11处理HepG2-HBx细胞毒性及细胞凋亡率均低于HepG2-3.1及HepG2。 结论： HBx可以抑制Fas通路介导的肝细胞凋亡。.
Keywords: Apoptosis; Fas; HepG2 cells; Hepatitis B virus X gene; Hepatocytes.