中国免疫学杂志2011，Vol.27Issue(3)：251-255,260,6.DOI:10.3969/j.issn.1000-484X.2011.03.014
幽门螺杆菌VacA N端片段通过线粒体途径诱导GES-1细胞凋亡
N-terminus of VacA derived from Helicobacter pylori induces GES-1 cells apoptosis through mitochondrial pathway
摘要
Abstract
Objective :This study was designated to investigate the vacuolating activity of N-terminus of vacA protein from Helicobacter pylori and the role of mitochondrial pathway in VacA-induced apoptosis in GES-1 cells .Methods :The recombinant eukaryotic expressing vector pDsRed-Monomer-C1/VacA encoding N-terminus of Helicobacter pylori VacA was constructed by our group previously .The plasmid was transfected into GES-1 cells ,and the vacuoles formation was assessed by electron microscope and neutral red uptake .Apoptosis was observed by Hoechst 33342 and Annexin V-FITC assay .Activation of Caspase-9 and Caspase-3 was detected by spectrophotometric method .Rho123 staining and ELISA were employed to define transmembrane potential and release of Cyt-C , respectively . Results : Recombinant plasmid pDsRedMonomer-C1/VacA could be expressed effectively in GES-1 cells .Vacuolar changes ,karyopyknosis and chromatin margination were observed after 24h post-transfection .The uptake of neutral red increased ,which had statistical significance in comparison with the control group ( P＜ 0.05 ) .Hoechst33342 stain showed obvious pycnosis .The apoptosis of GES-1 cells in recombinant transfection group was higher than that of the control group ,as assessed by increased binding of Annexin V-FITC ( p＜ 0 .05 ) .Spectrophotometric method showed that activation of Caspase-9 and Caspase-3 was time-dependent ,and peaks at 12 h and 24 h ,respectively .Rho123 staining indicated that the transmembrane potential decreased .ELLSA results demonstrated that the release of Cyt-C was time-dependent ,and the differences had statistical significance ( P＜ 0 .05 ) . Conclusion : H .pylori VacA N-terminal fragment could induce apoptosis and vacuoles formation of GES-1 cells .VacA could induce activation of Caspase-9 and Caspase-3 ,and mitochondrial pathway is probably involved in VacA-induced apoptosis in GES-1 .关键词
幽门螺杆菌/VacA/GES-1细胞/线粒体途径/细胞凋亡分类
医药卫生引用本文复制引用
杨卓,张艳,黎村艳,于文,曹斌,余敏君..幽门螺杆菌VacA N端片段通过线粒体途径诱导GES-1细胞凋亡[J].中国免疫学杂志,2011,27(3):251-255,260,6.基金项目
本文为湖南省自然科学基金(06JJ2093)、湖南省教育厅青年基金课题(08B067)和湖南省高校"病原体致病机制与防治"科技创新团队资助项目(湘教通[2010]212号) （06JJ2093）
