Effect Of Helicobacter Pylori Components On Proliferation And Apoptosis Of Gastric Epithelial Cell

ObjectiveTo observe the in vitro effects of Helicobacter pylori (Hp)components including secretory component,cell membrane,cytoplasm andHp lipopolysaccharide on proliferation and apoptosis of gastricepithelial cells. To contribute to searching for Hp component onproliferation of gastric epithelial cells.Methods1)The components(secretory component,cell membrane,cytoplasm)of cagA~+Hp (international standard strain NCTC11637)obtained with anultrasonicator and protein amounts in components were measured byBradford-based assay. H-LPS and LPS of Escherichia coli (E-LPS) wereextracted by phenol-water method.The H-LPS and E-LPS preparations weredetected by turbidimetry assay. SGC-7901 cells were cocultured withcagA~+Hp components at different protein concentrations(10-0.05ug/ml) orH-LPS at different doses(5-120 EU/ml). The effects of Hp components onSGC-7901 cell line such as morphologic change,proliferation and apoptosiswere percieved by fluorescence straining,MTT colorimstric methods andDNA-fragment assay;2)We extracted cell membrane component of cagA~-Hp(NCTC11639)and Escherichia coli. The growth of SGC-7901 cell line wasobserved using coincubation of Escherichia coli or Hp membrane by MTTcolorimstric methods;3)CagA~+Hp membrane proteins of variale solubilitywere extracted by the P-PEK procedure for gram-negative bacteria.SGC-7901cells were cocultured with membrane proteins of variale solubility aloneor combination at different concentrations. The growth rate of cell wasevaluated by MTT colorimstric methods and Ki-67 immunohistochemicaltechnique.Results (1)Results of the proliferation effect of cagA+Hp components onSGC-7901 cells : The MTT values of SGC-7901 cells were decreased with theincreasing concentration of cagA+Hp secretory component orcytoplasm.Membrane proteins enhanced proliferation at low doses(≤1ug/ml), while higher doses(≥2.5ug/ml) of cagA+Hp membrane proteinsresulted in growth arrest; H-LPS inhibited cell proliferation at highdoses(≥15 EU/ml). Hp membrane proteins of variale solubility inhibitedcell proliferation alone,while combinational membrane proteins( Proteins with intermediate solubility added to insolubleproteins,fraction2-4) from cagA+ Hp enhanced proliferation at low doses(p﹤0.05). Immunocytochemical result of Ki-67 expression in SGC-7901cell showed that Ki-67 labelling index(LI)at the group of low dose offraction2-4 was significantly higher than that at control group(p﹤0.05). (2)Results of the effect of Hp and E.coli membrane proteins onSGC-7901 cells: The SGC-7901 cell were cultured for 24h in vitro beforeproteins was added directly into the culturing medium.The growth ofSGC-7901 cells was promoted with the cocultured cagA+ and cagA-Hp membraneat low doses of 0.1 or 0.2ug/ml, While the proliferation rate of cellsin the result of cagA+Hp membrane was higher than that of cagA-Hp membrane(p﹤0.05); (3)Results of the apoptosis effect of cagA+Hp componentson SGC-7901 cells : The SGC-7901 cells incubated with Hp componentsexhibited some typical apoptosis-specific morphological features underthe fluorescence microscope. Especially on the role of Hp membranecomponent the morphology of SGC-7901 cells was changed most: they showedbigger volume, vacuole in cytoplasm,cell membrane budding and'cellshrinkage. However, no obvious morphology change were observed in controlgroup; The DNA-fragment by agarose gel electrophoresis demonstrated thetypical ladder pattern on the SGC-7901 cells directly exposed to Hpcomponents.Conclusions CagA+Hp secretory component,cytoplasm ,H-LPS and high doses of Hpmembrane components can inhibited proliferation and induce apoptosis invitro alone;.Both cagA+Hp and cagA-Hp membrane components can promoteproliferation at low doses;With the extract of Hp membrane proteins ofvariale solubility ,we found that combinational membrane proteins(fraction2-4) from cagA+ Hp enhanced proliferation at low doses(p﹤0.05).