乙型肝炎病毒全X基因对HEK293细胞功能的影响

目的克隆并构建乙型肝炎病毒(HBV)全X基因表达载体,探讨HBV全X基因对HEK293细胞功能的影响,并比较HBV全X基因与X基因对细胞影响的差异。方法应用基因克隆的方法从慢性HBV感染者血清中获得HBV全X及X基因,构建带有HBV全X基因与X基因的真核表达载体以及带有绿色荧光基团GFP的重组质粒;细胞转染技术将其分别转入HEK293细胞,RT-PCR及Western-blot技术验证HBV全X基因及X基因在细胞内的表达;荧光显微镜下观察其在细胞内的定位情况;MTT法检测细胞增殖率的变化;Annexin V流式细胞分析技术检测其对细胞凋亡率的影响;TRAP实时荧光定量PCR法检测各组细胞端粒酶活性的变化。结果成功构建带有HBV全X基因的重组质粒且目的基因能在HEK293细胞内高表达;表达的HBV全X蛋白主要分布在胞核,而X蛋白在胞质、胞核均有分布;转染HBV全X和X基因后细胞增殖率分别为(0.826±0.002)%和(0.805±0.013)%,显著高于转染空质粒(0.691±0.035)%和未转染对照组(0.681±0.018)%,差异具有统计学意义(P<0.05);转染HBV全X和X基因后细胞凋亡率分别为(25.25±3.02)%及(21.34±2.16)%,显著高于转染空质粒(13.49±1.49)%及未转染细胞对照组(11.37±1.58)%,差异具有统计学意义(P<0.05);转染全X及X基因细胞的端粒酶活性亦显著高于对照组,差异有统计学意义(P<0.05);全X转染组细胞的增殖率、凋亡率及端粒酶活性均高于X转染组,但差异均无统计学意义(P>0.05)。结论 HBV全X基因具有促细胞增殖、凋亡以及影响细胞端粒酶活性的作用,其可能通过不同于HBV X基因的细胞内途径影响细胞的生物学功能,从而在HBV相关的原发性肝细胞癌发生发展中起作用。

Effect of hepatitis B virus whole-X gene on HEK293 cells

Objective To clone and construct hepatitis B virus(HBV) whole-X(WX) gene eukaryotic expression vectors and investigate the effect of HBV WX gene on HEK293 cells by using HBV X gene as a control. Methods Eukaryotic expression vector pcDNA3.1-WX and pcDNA3.1-X and recombinant plasmids pEGFP-WX and pEGFP-X were constructed by gene cloning technologies.After recombinant plasmid was transfected into HEK293 cells,the gene expression was determined by RT-PCR and Western blot assays,and the subcellular localization of expressed EGFP-WX and EGFP-X proteins was observed by fluorescence microscope.We detected the changes of cell proliferation,apoptosis and telomerase activities after HBV WX and X gene were transfected into HEK293 cells by MTT,Annexin V flow cytometry analysis and TRAP real-time-PCR methods,respectively. Results HBV WX and X gene eukaryon expression vectors and recombinant plasmids were constructed successfully and the genes could be expressed in HEK293 cells.HBV WX protein was mainly located in the cell nucleus,but X protein was located in the cytoplasm and nucleus of HEK293 cells.After WX and X genes were transfected into HEK293 cells,cell proliferation capacity in WX and X groups was(0.826±0.002)% and(0.805±0.013)%,respectively,which was significantly higher than that in pcDNA3.1 and HEK293 cell control groups (0.691±0.035)% and(0.681±0.018)%,P<0.05].Cell apoptosis rate in WX and X groups was(25.25±3.02)% and (21.34±2.16)%,which was significantly higher than that in pcDNA3.1 and HEK293 cell control groups (13.49±1.49)% and(11.37±1.58)%,P<0.05].The level of telomerase activities in WX-and X-transfected gene groups was also significantly higher than that in control groups(P<0.05).Notably,cell proliferation capacity,apoptosis rate and telomerase activities of HBV WX-transfected gene were all higher than those of X-transfected gene although these differences were no significant(P>0.05). Conclusion HBV WX gene has stronger functions than HBV X gene in promoting cell proliferation,inducing cell apoptosis and increasing cell activities of telomerase.Thus,HBV WX gene may play a role in HCC progression via a different cellular pathway from HBV X gene.