HBV X基因在HL-7702肝细胞中的表达促进细胞凋亡并上调HSP70基因的表达

The Expression of HBV X Gene in HL-7702 Cells can Promote Cell Apoptosis and Up-regulate the Expression of HSP70 Gene

目的研究乙型肝炎病毒X(HBV X)基因在肝细胞HL-7702中的稳定表达对其细胞凋亡和表达HSP70的影响,并探讨二者之间的关联。方法将已构建好的HBV X基因真核表达载体pcDNA3-X转染人肝细胞HL-7702,48h后经G418筛选出稳定表达HBV X基因的肝细胞株(L02/HBx)。RT-PCR、蛋白印迹实验鉴定L02/HBx细胞HBV X基因的稳定表达。以转染空质粒肝细胞(L02/pcDNA3)为对照,运用流式细胞分析、TUNEL分析和DNA ladder观察检测L02/HBx细胞凋亡情况。基因芯片技术筛查L02/HBx和L02/pcDNA3两组肝细胞差异表达的凋亡相关基因,并运用蛋白印迹实验技术对差异表达的HSP70基因进行蛋白水平验证。结果 RT-PCR和蛋白印迹实验显示,L02/HBx细胞中有HBV X基因mRNA和蛋白的表达。流式细胞和TUNEL分析显示,L02/HBx细胞组的凋亡率显著高于对照组L02/pcDNA3,DNA ladder可观测到L02/HBx的凋亡现象,而对照组未观测到。两次基因芯片结果筛查出L02/HBx细胞组与对照组差异表达基因HSP70,蛋白印迹实验进一步证实HSP70在L02/HBx细胞中的蛋白表达显著高于未表达X基因的肝细胞组。结论 HBV X基因在肝细胞HL-7702中的表达促进了肝细胞的凋亡,上调了肝细胞中HSP70基因的表达,从而在乙型肝炎病毒致肝细胞癌变的过程中起重要作用。

Objective To investigate the effects of hepatitis Bvirus X （HBVX ） gene expression in HL-7702 live cells on cell apoptosis and the expression of HSP70 gene ,and explore their association . Methods The eukaryotic expression vector of HBVX gene （pcDNA3-X） was transfected into human liv-er cell HL-7702 and selected with G418 to construct a new cell line L02/HBx ,which was confirmed to ex-press HBVX gene steadily by RT-PCR and western-blot analysis . Compared with HL-7702 cells trans-fected with empty vector （L02/pcDNA3） ,apoptosis of L02/HBx were detected by flow cytometric analy-sis ,TUNEL analysis and DNA ladder . Then gene arrays were used to select out the different apoptosis gene expressions between the two cells . Then the differential expression of HSP70 was further confirmed by Western-blot analysis . Results RT-PCR and Western-blot analysis showed L02/HBx cells canex-press mRNA and protein of HBVX gene steadily . Flow cytometric and TUNEL analysis showed that the apoptosis rates of the L02/HBx cells were much higher than those of L02/pcDNA3 . And apoptosis phenomena in L02/HBx were observed by DNA ladder ,but not in L02/pcDNA3 . HSP70 ,as a differen-tial expression gene in the two cells ,was selected out by gene array . Western-blot analysis further con-firmed that the protein expression of HSP70 in L02/HBx cells was much higher than that in HL-7702 cells without expressing X gene . Conclusion The expression of HBVX gene in HL-7702 cells can promote the apoptosis of live cells and up-regulate the expression of HSP70 gene ,which play important roles in the process of HBV-induced HCC .