丙型肝炎病毒核心区蛋白对 HepG2细胞周期、细胞凋亡和细胞端粒酶活性的影响

目的　研究丙型肝炎病毒核心区 (HCV C)蛋白对肝癌细胞HepG2细胞周期、细胞凋亡和细胞端粒酶活性的影响。方法　首先运用基因重组技术构建含有HCV C基因的真核表达质粒pcDNA3.1( ) ,然后利用脂质体介导将重组真核表达质粒转染HepG2 ,经G4 18筛选获得稳定转染HepG2细胞 (HCV C转染HepG2细胞 ) ,经逆转录 聚合酶链反应技术 (RT PCR)和间接免疫荧光法证实其中有HCV C蛋白表达。然后进行如下实验 :( 1)利用四甲基偶氮唑蓝比色 (MTT)法检测HCV C转染HepG2细胞、空白质粒转染HepG2细胞和未转染HepG2细胞的生长增殖率 ;经流式细胞术(FACS)检测 3组细胞的细胞周期 ;( 2 )经流式细胞术检测细胞凋亡率 ;( 3)经端粒重复扩增 酶联免疫吸附试验 (TRAP ELISA)法检测上述 3组细胞端粒酶活性表达情况。结果　 ( 1)HCV C转染HepG2细胞增殖率显著高于空白质粒转染HepG2和未转染HepG2细胞增殖率 ;HCV C转染HepG2细胞S期所占百分率高于未转染HepG2细胞S期所占百分率 ;( 2 )HCV C转染HepG2细胞凋亡率显著低于无HCV C转染细胞凋亡率 ;( 3)上述 3组细胞端粒酶活性之间差异无显著性。结论　 ( 1)HCV C蛋白具有抑制细胞凋亡的作用 ;( 2 )HCV C蛋白促进HepG2从G0 /1期进入S期 ,从而可能促进细胞生长增殖 ,抑制细胞凋亡 ;( 3)HCV

The influence of HCV core protein on cell apoptosis, cell cycles and cell telomerase activities of HepG2 cells

Objective To investigate the influence of HCV core protein on cell apoptosis, cell cycles and cell telomerase activities of HepG2 cells. Methods A eukaryotic expression plasmid containing HCV C gene was constructed by DNA recombinant technique and the recombinant plasmid was transfected into HepG2. Thereafter, HepG2 cells transfected with recombinant eukaryotic expression plasmid were obtained. The HCV C mRNA and protein in HepG2 cells transfected with recombinant plasmid were verified by RT PCR and indirect immunofluorescence assay. The HepG2 cells were studied as follows: (1) The cell proliferation ratio of three groups cells(HepG2 cells transfected with the recombinant plasmid, HepG2 cells transfected with blank plasmid and HepG2 cells without transfection) was evaluated by MTT assay; the cell cycles were also examined by FACS. (2) The apoptotic ratio of three groups cells were examined by FACS. (3) The cell telomerase activities of all three group cells were examined by TRAP ELISA assay. Results (1) The cell proliferation ratio in the group of HepG2 cells transfected with recombinant plasmid was higher than that of the group of HepG2 cells transfected with blank plasmid or the group of HepG2 cells without transfection; The proportion of phase S in the group of HepG2 transfected with the recombinant plasmid was significantly higher than that of the group of HepG2 without transfection. (2) The apoptotic ratio in the group of HepG2 cells transfected with recombinant plasmid was significantly lower than that of the group of HepG2 cells transfected with blank plasmid or the group of HepG2 cells without transfection. (3) There were no significant differences among the three group cell telomerase activities. Conclusions (1) HCV C protein had the potential role in inhibiting cell apoptosis. (2)HCV C protein could induce HepG2 cells from phase G 0/1 to phase S, and might promote cell proliferation, inhibit cell apoptosis. (3) HCV C protein had no influence on cell telomerase activities of HepG2 cells, thus HCV C protein regulated cell cycle, promoted cell proliferation and inhibited cell apoptosis not by enhancing cell telomerase activities.