The Role Of Mitchondrial Pathway In Apoptosis Of Ges-1 Cell Induce By Helicobacter Pylori VacA

Objective:This study was designated to investigate the vacuolating activity of N-terminus gene of vacA gene from Helicobacter pylori and the role of mitochondrial pathway in VacA-induced apoptosis in GES-1 cells.Methods:The N-terminus of eukaryotic expression vector pDsRed-Monomer-C1/VacA constructed by our group previously was transfected into GES-1 cells, the expression of VacA was detected by Western-blot, and the vacuolar changes was assessed by electron microscope and neutral red uptake. Apoptosis was observed by Hoechst 33342/Annexin V-FITC assay and electron microscope, 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 activation of Caspase-9 and Caspase-3 was time-dependent, and peaks at 12h and 24h, respectively. Rho123 staining indicated the transmembrane potential decreased, as compared with control group(P<0.05). ELISA results demonstrated release of Cyt-C was time-dependent, the differenes had statistical significance (P<0.05).Conclusions:1. The instantaneous expression of VacA could induce GES-1 cells apoptosis and vacuolar changes.2. VacA could induces activation of Caspase-9 and Caspase-3, and mitochondrial pathway is probably involved in VacA-induced apoptosis in GES-1.