乙型肝炎病毒x影响肝细胞增殖与凋亡的初步研究

The initial research on the influence of HBx on the proliferation and apoptosis of hepatocytes

目的观察乙型肝炎病毒x(HBx)导入正常肝细胞后对细胞周期及凋亡的影响,探讨HBx致肝细胞癌的发生机制。方法应用脂质体转染重组质粒pcDNA3.1(+)/v5-hisB-HBx后,G418筛选获得阳性克隆L02/HBx,分别用RT-PCR和Westernblot鉴定其HBxmRNA与蛋白的表达。噻唑蓝比色法(MTT)及细胞生长曲线观察细胞增殖情况,流式细胞术分析其细胞周期特征与凋亡率。结果构建的L02/HBx细胞中可稳定表达HBxmRNA及蛋白。与对照组比较,L02/HBx细胞增殖速度明显加快(P<0.05);其G1期细胞比例明显减少,S期细胞比例显著增加,凋亡率明显降低(P<0.05)。结论成功构建稳定表达HBx的转基因细胞株L02/HBx,初步显示HBx可促进肝细胞增殖,抑制细胞凋亡,从而加速细胞周期进程,可能参与肝细胞的恶性转化。

Objective Observe the influence of HBx inducted into normal hepatocytes on the cell cycle and apoptosis to study the mechanism of the carcinogenesis of hepatoma induced by HBx. Methods Recombinant plasmid pcDNA3.1 （ ＋ ）/v5-hisB-HBx was transfected into L02 cell line by liposome. The positive clone was screened out by G418. HBx mRNA and the target protein were identified by RT-PCR and Western blotting respectively. Cell proliferation was assayed by MTT and cell growth curve analysis. Life cycle and apoptosis were analysed by flow cytometry. Results The expression of HBx mRNA and HBx protein was confirmed in L02/HBx cell line. The proliferation of LO2/HBx significantly accelerated compared with control（P〈0.05）. The proportion of cells in G1 stage decreased with cells in S stage increased,the apoptosis of which decreased significant compared with control （ P 〈 0.05 ）. Conclusions The transgenic cell line L02/HBx stably expressing HBx was constructed successfully. HBx could promote the proliferation of hepatocytes,inhibit the apoptosis and quicken the life cycle proceeding, which may take a part in the malignant transformation of hepatocytes.