Effect Of Virulence Factors From Helicobacter Pylori On Proliferation And Apoptosis Of Gastric Epithelial Cell

Objective To investigation the effect of four virulence factors CagA, OipA, UreB, UreC from Helicobacter pylori (HP) on proliferation and apoptosis of gastric epithelial cell.To contribute to searching for carcinogenic components of HP. To offer security index for developing DNA vaccine of four virulence factors.Methods The whole sequences of cagA,ureB were amplified by RT-PCR from standard strain NCTC11637, inserted into pGEM-T Easy vector and sequenced.The ORFs of four genes from recombinant T vectors of cagA,ureB and recombinant T vectors of oipA,ureC which were cloned before with the same method were inserted directionally into the eukaryotic expression vector pcDNA3.1, SGC-7901 cells were transfected with four eukaryotic recombinant plasmids and pcDNA3.1 by Lipofectamine2000. The cloned cells resisting Hygromacine were picked out. Gene expression of four positive clones were detected by RT-PCR. Expression of OipA,UreC were detected respectively with chicken anti-OipA antibody and chicken anti- UreC antibody by immune-blot. Effects of expression of four genes on cell phenotype,proliferation,apoptosis and cell cycles were detected by fluorescence straining,MTT assay and flow cytometyr.Results The four eukaryotic recombinant plasmids pccagA,pcureB,pcoipA,pcureC and five cloned cells SpcDNA3.1,ScagA,SureB,SoipA,SureC containing corresponding recombinant plasmids were constructed successfully. Corresponding gene fragments in cloned cells were amplified by RT-PCR, OipA and UreC expression in SoipA and SureC were identified with corresponding antibodies by immune-blot. The morphology of cloned cells were observed under the fluorescence microscope: The ScagA changed greatestly of all cells, it showed bigger volume, vacuole in cytoplasm, cell membrane budding and'cell shrinkage. A few SureB also showed cell membrane budding and'cell shrinkage. However, no obvious morphology change were observed in SoipA and SureC. Results of MTT assay: There was no statistic significance(P>0.05) between the growth of SpcDNA3.1 and that of SureB,SoipA and SureC. But the growth of ScagA was more slowly than SpcDNA3.1 (P=0.009) showed that the growth of ScagA was inhibited. The apoptosis rate of SpcDNA3.1,ScagA,SureB,SoipA,SureC detected by FCM were 1.44(±0.08),9.23(±0.87),3.78(±0.23),3.95(±1.73),3.73(±0.83), respectively. The apoptosis rate of ScagA and SureB were higher than that of SpcDNA3.1(The value of P respectively : 0.02,0.007). There was no statistic significance(P>0.05) between the apoptosis rate of SpcDNA3.1 and that of SoipA and SureC. Results of cell cycle distribution of ScagA and SureB showed that propotion of ScagA and SureB cells in S phase was increased, propotions of ScagA and SureB cells in G2/M and G0/G1 phase were all decreased .Conclusion Expression of cagA in SGC-7901 changed cell phenotype, inhibited cell proliferation and increased cell apoptosis in vitro. Expression of ureB in cell increased cell apoptosis. UreB, one of urease sub-units, induced cell apoptosis alone. Expression of ureC and oipA had no effect on cell function, so they could be prepared for DNA vaccine.