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

作　　者：杨卓[1] 张艳[1] 黎村艳[1] 于文[1] 曹斌[1] 余敏君[1] 

机构地区：[1]南华大学医学院病原生物学研究所,衡阳421001

出　　处：《中国免疫学杂志》2011年第3期251-255,260,共6页Chinese Journal of Immunology

基　　金：湖南省自然科学基金(06JJ2093);湖南省教育厅青年基金课题(08B067);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)

摘　　要：目的:本研究初步探讨幽门螺杆菌(H.pylori)空泡毒素单一毒力决定簇对GES-1胃黏膜上皮细胞凋亡的影响,为进一步研究H.pylori的致病机制奠定基础。方法:将本课题组已构建的pDsRed-Monomer-C1/VacA N端真核表达载体转染至GES-1细胞中,Western blot鉴定VacA蛋白在细胞中的表达;电子显微镜和中性红摄取试验检测GES-1细胞的空泡样变;Hoechst33342染色、Annexin V-FITC凋亡检测试剂盒以及电镜观察细胞凋亡情况,同时采用分光光度法检测细胞Caspase-9、Caspase-3的活化程度,Rho123检测细胞跨膜电位的变化,ELISA法检测细胞色素C的释放。结果:重组质粒pDsRed-Monomer-C1/VacA转染GES-1细胞24小时后,电镜下可明显观察到空泡样变及核固缩、染色质边聚等凋亡特征,重组质粒组部分细胞发生空泡样变;Hoechst 33342染色后镜下可见明显的核染色质浓缩,Annexin V-FITC凋亡检测试剂盒检测发现重组质粒组细胞凋亡率明显高于对照组(P<0.05);Caspase-9和Caspase-3活化程度呈时间依赖性增加,分别在12小时和24小时达到峰值;Rho123染色发现线粒体跨膜电位明显降低;重组质粒转染组释放细胞色素C的浓度呈时间依赖性正相关,与空质粒组及对照组相比差异显著(P<0.05)。结论:VacA N端片段可诱导GES-1细胞凋亡及空泡样变,VacA蛋白可激活Caspase-9和Caspase-3,且可能主要通过线粒体途径诱导GES-1细胞凋亡。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 pDsRed-Monomer-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.ELISA 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 号：R392.32[医药卫生—免疫学]