乙型肝炎病毒X蛋白稳定表达株的构建及沉默效果观察

摘要

Objective To construct the HepG2-HBX cell strain that stably expressing the hepatitis B virus protein X (HBX) and to explore the specific silencing effect of siRNA on the HBX gene. Methods The pCDNA3. 1 ( + )/HBX-Flag plasmid containing the HBX sequence was transfected into the HepG2 cell lines with lipofectamine 2000,and the HepG2-HBX cell line stably expressing the HBX gene was screened out with G418; RT-PCR and immunohistochemistry were employed to validate the expression of the HBX gene; the siHBX fragment targeting the HBX gene was chemically synthesized and transfected in to the HepG2-HBX cell line, and RT-PCR, real time quantitative RT-PCR and Western blot were respectively performed to evaluate the the expression of HBX at both the mRNA and protein level. The cell cycle of HBX19 was examined by flow cytometry. Results The HepG2-HBX cell strain stably expressing the HBX gene was successfully constructed and the siHBX fragment may specifically inhibit HBX expression and cell cycle progression. Conclusion siHBX is a potential targeted strategy to inhibit the HBX gene in liver cancer cells.%目的 构建稳定表达乙型肝炎病毒X蛋白(HBX)基因的HepG2-HBX细胞系,观察小分子干扰RNA(siRNA)对HBX基因的特异性抑制效果.方法 用脂质体将含有HBX序列的真核表达质粒pCDNA3.1(+)/HBX-Flag转染人HepG2细胞系,通过G418筛选后,分别经RT-PCR和免疫组化对HBX的表达进行验证;化学合成靶向HBX的siRNA(即siHBX),转染入HBX稳定表达株,分别以RT-PCB和实时荧光定量RT-PCR检测HBX mRNA表达水平,Western blot检测HBX蛋白表达水平以验证siHBX对HBX基因的沉默效应,流式细胞仪检测细胞周期变化.结果 成功构建表达HBX基因的HepG2-HBX细胞系;siHBX可以高效特异抑制HBX表达,并抑制细胞周期进展.结论 siHBX可作为一种靶向抑制肝癌细胞系内HBX表达的策略,为后续HBV感染相关性肝癌的治疗奠定了实验基础.