丙型肝炎病毒核心蛋白转染HepG2细胞对其p53表达的影响

Effect of HCV core protein transfected into HepG2 cells on p53 activities

目的研究丙型肝炎病毒核心蛋白(HCV-core protein,HCV-C)对HepG2细胞周期、细胞凋亡和p53蛋白表达的影响。方法表达HCV-C的真核表达质粒pcDNA3.1-core,通过Lipofectamine基因转染法转染HepG2细胞,经G418筛选获得稳定转染HepG2细胞,经Western Blot证实HCV核心蛋白阳性表达。利用四甲基偶氮唑蓝比色(MTT)法,平板克隆实验和流式细胞术,蛋白印迹和免疫细胞化学法检测HCV核心蛋白对细胞生长增殖率、细胞周期、细胞凋亡率和p53蛋白表达的影响。结果HCV-C转染组细胞增殖率显著高于空白质粒转染组和未转染组;HCV-C转染组细胞S期所占百分率高于未转染组;HCV-C转染组细胞凋亡率显著低于未转染组;HCV-C转染组细胞突变型p53蛋白的表达高于空白质粒转染组和未转染组。结论HCV核心蛋白可能通过促进突变型p53蛋白的表达,促进HepG2从G0/1期进入S期,促进细胞生长增殖,抑制细胞凋亡。

Objective To investigate the influence of HCV core protein on apoptosis, cell cycles and p53 activities of HepG2 cells. Methods A cell line expressing stably HCV core protein was constructed by transfecting the plasmid pcDNA3.1-core（expressing HCV core protein） into HepG2. HepG2 cells transfected with recombinant eukaryotic expression plasmid were obtained. The HCV-C protein in HepG2 cells transfected with recombinant plasmid was verified by Western Blot assay. The cell proliferation of three groups cells： HepG2 cells transfected with the recombinant plasmid, HepG2 cells transfected with blank plasmid and HepG2 cells without transfection were evaluated by MTT assay ; the cell cycles and apoptotic ratio of different groups were also examined by FACS. The levels of expression of p53 protein of different groups were examined by immunocytochemical method. Results 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 without transfection. The apoptotic ratio in the group of HepG2 cells transfected with recombinant plasmid was significantly lower than those of the group without transfection. The expression ratio of mutation p53 protein in the group of HepG2 cells transfected with recombinant plasmid was significantly higher than those of the other groups. Conclusion HCV-C protein could induce HepG2 cells from phase G0/GI to phase S, promote cell proliferation and inhibit cell apoptosis by promoting the expression ratio of p53 protein mutation.