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

作　　者：陈红英[1] 陈治新[1] 王小众[1] 

机构地区：[1]福建医科大学附属协和医院消化内科,福州350001

出　　处：《福建医科大学学报》2013年第5期265-270,共6页Journal of Fujian Medical University

基　　金：教育部高校博士点专项基金资助项目(20093518110003);福建省科技人才基金资助项目(2008F3043);福建省临床医学重点专科资助项目;福建省卫生厅青年基金立项医院基金资助项目(XH200801)

摘　　要：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 .

关 键 词：肝细胞 肝炎病毒  乙型 基因  病毒 基因表达 细胞凋亡 细胞  培养的 癌  肝细胞 HSC70热 休克蛋白质类 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R329.25[医药卫生—基础医学]