乙型肝炎病毒表面抗原大蛋白致HepG2细胞凋亡的蛋白质组学分析  

Proteomics analysis of the apoptotic effects of hepatitis B virus large envelope protein on HepG2 cells

作　　者：郑大利[1] 黄清玲[1] 吴云丽[1] 林建银[1] 林旭[1] 

机构地区：[1]福建医科大学省部共建消化道恶性肿瘤教育部重点实验室福建省肿瘤微生物学重点实验室,福州350004

出　　处：《中华微生物学和免疫学杂志》2010年第12期1085-1091,共7页Chinese Journal of Microbiology and Immunology

基　　金：福建省教育厅资助省属高校项目（2007F5050）;福建省杰出青年科学基金项目（2009J06016）;福建省高等学校科技创新团队培育计划基金（FMU-RT001）

摘　　要：目的 研究乙型肝炎病毒（hepatitits B virus,HBV）表面抗原大蛋白（large envelope protein,LHB）对HepG2细胞凋亡的影响,并以蛋白质组学方法探讨其机制.方法 以腺病毒表达载体pShuttle-IRES-hrGFP-1克隆HBV LHB基因,经重组、包装后获得重组腺病毒Ad-LHB,与空载体重组腺病毒Ad-GFP分别感染HepG2细胞,采用Annexin V-FITC细胞凋亡检测试剂盒、JC-1细胞凋亡线粒体膜电位检测试剂盒和碘化丙啶（PI）染色检测细胞凋亡情况.Ad-LHB及Ad-GFP分别感染HepG2细胞,提取细胞总蛋白,各取600μg蛋白样品进行双相电泳,R350染色,以双相电泳图像分析软件ImageMaster 2D Platinum确定差异蛋白点,Ettan斑点自动挖胶系统切取差异蛋白,脱色酶解后进行MALDI-TOF-TOF MS鉴定并分析.结果 Ad-LHB感染导致HepG2细胞凋亡明显增加.双向电泳及扫描分析显示Ad-LHB与Ad-GFP感染的HepG2细胞共有39个蛋白差异.质谱分析并确定其中的33种共36个蛋白,这些差异蛋白质有9种与细胞凋亡密切相关,其中,CAPN2、eIF3K和PPP2CB在Ad-LHB感染HepG2细胞表达增高,SERPINH1、LASP1、PRDX1、DHRS2、LDHA和PSMA4在Ad-LHB感染HepG2细胞表达降低.结论 HBV LHB可致HepG2细胞凋亡,多种细胞凋亡相关蛋白参与此过程.Objective To assess the effects of hepatitis B virus（HBV） large envelope protein （LHB） on the apoptosis of HepG2 cells and explore the possible mechanism by proteomic approaches.Methods LHB gene was cloned into pShuttle-IRES-hrGFP-1,and the recombinant adenovirus either barboring LHB（Ad-LHB） or empty vector（Ad-GFP） were separately generated.Annexin V-FITC apoptosis detection kit,JC-1 mitochondrial membrane potential assay kit and propidium iodide（PI） staining kit were employed combined with flow cytometry to detect the apoptotic cells infected with Ad-LHB or control of Ad-GFP.The cellular proteins were collected after infection of HepG2 cells by Ad-LHBs or Ad-GFP,and a total of 600 μg proteins were submitted to two-dimensional gel electrophoresis（2-DE） and stained with R350.The gel images were captured by ImageScanner Ⅱ Imaging System,the differentially expressed proteins were identified by ImageMaster 2D Platinum analysis software and picked up by Ettan Spot Picker.After enzyme digestion,the protein samples were analyzed by MALDI-TOF-TOF MS.Results HepG2 cells infected with Ad-LHB were much more prone to apoptosis.There were thirty nine differentially expressed proteins were determined by 2-DE between HepG2 cells infected with Ad-LHB and Ad-GFP,and they were identified ultimately and categorized into thirty three kinds of proteins by MALDI-TOF-TOF MS.Among these proteins,nine were found to be closely related to cell apoptosis,in which CAPN2,eIF3K and PPP2CB were higher expressed in Ad-LHB infected HepG2 cells,and SERPINH1,LASP1,PRDX1,DHRS2,LDHA and PS-MA4 were lower expressed in Ad-LHB infected HepG2 cells.Conclusion LHB could induce apoptosis of HepG2 cells,and several apoptosis-related proteins participated in this process.

关 键 词：乙型肝炎病毒 蛋白质组学 双向凝胶电泳 肝细胞 细胞凋亡 

分 类 号：R51[医药卫生—内科学]