| Objective: Cytotoxic associated antigen A(Cag A) is an important virulence factor of Helicobacter pylori(Hp), it is closely related to the occurrence of gastric adenocarcinoma. It had been certified that there is an interaction between Cag A and YWHAE in gastric mucosal epithelial cell, so the study used cag A mutants as contrasted with wild-type strains to verify the impact on cells caused by Cag A. The aim of this paper was to investigate the effect that induced by the interaction between Cag A and YWHAE on cell migration in gastric mucosal epithelial, then further elucidate the function of Cag A in the pathogenesis of Hp.Methods1. The verification of Cag A and YWHAE in cell positioning: Immunofluorescence was processed by using the specific antibody of Cag A and YWHAE on AGS or SGC7901 which were infected by Hp NCTC 11637. The cells were then investigated the localization of Cag A and YWHAE by the laser scanning confocal microscopy.2. The construction of NCTC 11637 cag A mutant—NCTC 11637Δcag A: Based on the theory of homologous recombination, the isogenic cag A knock-out mutant vector which had been inserted a kanamycin resistance gene cassette was constructed by designed the primers of the upstream and downstream homologous arms of cag A according to NCTC 11637 cag A gene. The mutant vector was transferred into Hp NCTC 11637 by electroporation and the mutant strains were selected from selective Columbia agar plates. The genomic DNA of the transformants was extracted for the PCR identification of the cag A gene and km R, the protein of the transformants were extracted for the Western blot identification of Cag A. The same identification had been done after 25 generations culture of the transformants to identify the genetic stability of cag A gene deletion.3. The effect of NCTC 11637 Cag A on gastric epithelial cell: Co-culture Hp(NCTC11637 or NCTC 11637Δcag A) with SGC7901 at a multiplicity of infection(MOI) of100, using the following techniques to investigate the effect of Cag A: MTS detection kit detected the rate of cell proliferation: continuous observation of 1-5d, cell proliferation rate = OD2-5d / OD1d; After 48 h infection, apoptosis and cell cycle were detected by flow cytometry; after 6h infection, the cells were undergoing the scratch wound-healing assay, and cells were photographed and measured by Carl Zeiss License Activation Tool right after wound-healing and 48 h afterwards. Cell migration distance=Width 6h-48 h.4. The over-expression and knock down of YWHAE in AGS and SGC7901: The plasmid p CMVTNT-YWHAE(our laboratory has been constructed)(or YWHAE si RNA) were transfected into AGS or SGC7901 via Lipofectamine 2000(or X-treme GENE si RNA) transfection reagent respectively. The over-expression and knock down of YWHAE were identified by Western blot after 48 h of transfection.5. The effect of the interaction between Cag A and YWHAE on cell migration: The plasmid p CMVTNT-cag A(our laboratory has been constructed) was transfected into AGS or SGC7901 via Lipofectamine 2000. The expression of Cag A was identified by Western blot after 48 h of transfection. Different concentrations of p CMVTNT-cag A or p CMVTNT-YWHAE were transfected into AGS or SGC7901, then scratch wound-healing assay was used to detect cell migration. The same method was used on AGS or SGC7901 which had been transfected proper concentration of p CMVTNT-cag A and p CMVTNT- YWHAE(or YWHAE si RNA) to detect cell migration.6. Statistical analysis: Data was analyzed by independent samples t test, single factor analysis of variance and factorial design ANOVA through SPSS20.0.Results1. By immunofluorescence co-localization, Cag A and YWHAE could be observed in the cytoplasm of AGS and SGC7901 at the same time, which indicated that Cag A and YWHAE can be co-located in the cytoplasm.2. After the identification by PCR, Western blot and multigenerational culture, NCTC11637Δcag A had been constructed successfully.3. The effects of NCTC 11637 Cag A on cell biological function(1) Cell proliferation: The cell proliferation rate of NCTC11637 group was more than NCTC11637Δcag A group from third day to fifth day, and there was a significant difference on third day and fifth day(P <0.05);(2) Cell apoptosis: Apoptosis rate of NCTC11637 group(1.82%) was less than NCTC11637Δcag A group(2.55%)(P <0.01);(3) Cell cycle: The G1, S, G2 phase cell percentages of NCTC11637 group were60.77%, 25.4%, 13.04%, and NCTC11637Δcag A group were 56.78%, 28.27 %,13.95%. There was a significant difference by the comparison between two groups in each period(P <0.05);(4) Cell migration: The cell migration distance of NCTC 11637group(147.39μm) was more than NCTC 11637Δcag A group(81.65μm)(P <0.05).4. Through Western bolt verification, the YWHAE protein expression in p CMVTNT-YWHAE group was more than p CMV-TNT group and YWHAE si RNA group was less than Negative Control group in AGS and SGC7901.5. Effect of interaction between Cag A and YWHAE on cell migration(1) Through Western bolt verification, the Cag A protein expression in transfected p CMVTNT- Cag A group was more than transfected p CMV-TNT group in AGS and SGC7901.(2) a. With the increase of p CMVTNT-cag A concentration, cell migration distance was increased in AGS and SGC7901, but with the increase of p CMVTNT-YWHAE concentration, cell migration distance gradually reduced. b.After co-transfected p CMVTNT-cag A and p CMVTNT-YWHAE in AGS and SGC7901, the tendency of cell migration distance between different groups was CMV-YWAHE< CMVTNT<Cag A+YWHAE<CMV-Cag A, and there was significant difference between the groups(P<0.05). c. After co-transfected p CMVTNT-cag A and YWHAE si RNA in AGS and SGC7901, the tendency of cell migration distance between different groups was CMV-YWAHE+NC <CMVTNT+YWHAE si RNA<Cag A+NC< Cag A+YWHAE si RNA, and there was significant difference between the groups(P<0.05).Conclusion1. The spatial location centified that there is an interaction between Cag A and YWHAE within the cell.2. NCTC 11637 Cag A could induce the cell proliferation, inhibit the apoptosis and arrest the cell cycle in the G1 phase.3. YWHAE could weaken the auxoaction of Cag A on cell migration. |
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