中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2010年
12期
1085-1091
,共7页
郑大利%黄清玲%吴云丽%林建银%林旭
鄭大利%黃清玲%吳雲麗%林建銀%林旭
정대리%황청령%오운려%림건은%림욱
乙型肝炎病毒%蛋白质组学%双向凝胶电泳%肝细胞%细胞凋亡
乙型肝炎病毒%蛋白質組學%雙嚮凝膠電泳%肝細胞%細胞凋亡
을형간염병독%단백질조학%쌍향응효전영%간세포%세포조망
Hepatitis B virus%Proteomics%Two-dimensional gel electrophoresis%Hepatocyte%Apoptosis
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.
目的 研究乙型肝炎病毒(hepatitits B virus,HBV)錶麵抗原大蛋白(large envelope protein,LHB)對HepG2細胞凋亡的影響,併以蛋白質組學方法探討其機製.方法 以腺病毒錶達載體pShuttle-IRES-hrGFP-1剋隆HBV LHB基因,經重組、包裝後穫得重組腺病毒Ad-LHB,與空載體重組腺病毒Ad-GFP分彆感染HepG2細胞,採用Annexin V-FITC細胞凋亡檢測試劑盒、JC-1細胞凋亡線粒體膜電位檢測試劑盒和碘化丙啶(PI)染色檢測細胞凋亡情況.Ad-LHB及Ad-GFP分彆感染HepG2細胞,提取細胞總蛋白,各取600μg蛋白樣品進行雙相電泳,R350染色,以雙相電泳圖像分析軟件ImageMaster 2D Platinum確定差異蛋白點,Ettan斑點自動挖膠繫統切取差異蛋白,脫色酶解後進行MALDI-TOF-TOF MS鑒定併分析.結果 Ad-LHB感染導緻HepG2細胞凋亡明顯增加.雙嚮電泳及掃描分析顯示Ad-LHB與Ad-GFP感染的HepG2細胞共有39箇蛋白差異.質譜分析併確定其中的33種共36箇蛋白,這些差異蛋白質有9種與細胞凋亡密切相關,其中,CAPN2、eIF3K和PPP2CB在Ad-LHB感染HepG2細胞錶達增高,SERPINH1、LASP1、PRDX1、DHRS2、LDHA和PSMA4在Ad-LHB感染HepG2細胞錶達降低.結論 HBV LHB可緻HepG2細胞凋亡,多種細胞凋亡相關蛋白參與此過程.
목적 연구을형간염병독(hepatitits B virus,HBV)표면항원대단백(large envelope protein,LHB)대HepG2세포조망적영향,병이단백질조학방법탐토기궤제.방법 이선병독표체재체pShuttle-IRES-hrGFP-1극륭HBV LHB기인,경중조、포장후획득중조선병독Ad-LHB,여공재체중조선병독Ad-GFP분별감염HepG2세포,채용Annexin V-FITC세포조망검측시제합、JC-1세포조망선립체막전위검측시제합화전화병정(PI)염색검측세포조망정황.Ad-LHB급Ad-GFP분별감염HepG2세포,제취세포총단백,각취600μg단백양품진행쌍상전영,R350염색,이쌍상전영도상분석연건ImageMaster 2D Platinum학정차이단백점,Ettan반점자동알효계통절취차이단백,탈색매해후진행MALDI-TOF-TOF MS감정병분석.결과 Ad-LHB감염도치HepG2세포조망명현증가.쌍향전영급소묘분석현시Ad-LHB여Ad-GFP감염적HepG2세포공유39개단백차이.질보분석병학정기중적33충공36개단백,저사차이단백질유9충여세포조망밀절상관,기중,CAPN2、eIF3K화PPP2CB재Ad-LHB감염HepG2세포표체증고,SERPINH1、LASP1、PRDX1、DHRS2、LDHA화PSMA4재Ad-LHB감염HepG2세포표체강저.결론 HBV LHB가치HepG2세포조망,다충세포조망상관단백삼여차과정.
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.
