The Function and Significance of Helicobacter Pylori Invading the Gastric Mucosa Epithelium
|Keywords||Helicobacter pylori（H. pylori） Invasiveness Digestive diseases AGS cells|
ObjectiveH. pylori (Helicobacter pylori, Hp) is a gram-negative microaerobion, long-termplanting in gastric mucosa. H. pylori infection is not only the pathogenic factorcausing atrophic gastritis, gastric ulcer and chronic inflammation, but also closelyrelated with malignant disease, such as gastric cancer and gastric mucosa associatedlymphoid tissue lymphoma. In recent years, a large number of research results showedHelicobacter pylori can affect on the stomach mucosa directly and reproduce withinthe epithelial cells, leading to the cell pathological changes. But the mechanism ofHelicobacter pylori invading the stomach epithelial cells is unknown, and theinteraction mechanism between Helicobacter pylori and the epithelial cells is also notclear. This research observed H. pylori isolated from different digestive diseasesinvade AGS cells and investigated the influence on AGS cells cellular functions, anddiscussed the relevance between Helicobacter pylori invasiveness and gastricpathopoiesia, which would contribute to exploring the pathogenesis mechanism of H.pylori.Methods1. H. pylori invade AGS cells1.1Using gentamicin invasion assays, helicobacter pylori standard strains(NCTC11637and SS1) and clinical strains (75plants, including from gastritis30plants, gastric ulcer25plants and gastric cancer20plants) infected AGS cells (MOI=50) for2h. After2h, infected epithelial cells were washed six times with culturemedium supplemented with100μg/ml gentamicin100μg/ml and then incubated withgentamicin-containing (25μg/ml) medium.Collect cells after infection4h and12h,then train on the helicobacter pylori blood plates. Colonies were grown and counted after6days of culture. Calculation and comparison the attack rate.1.2AGS cells were pulsed with the same bacterial strains at a multiplicity of infection(MOI) of50at37°C for2h and then the cells were washed four times with PBS.Collect cells and then train on the helicobacter pylori blood plates. Colonies weregrown and counted after6days of culture.This was the the number of bacterialadhesion.AGS cells infected for2h were washed six times with culture mediumsupplemented with100μg/ml gentamicin and then incubated with the same mediumas above for2h.Then colonies were grown and counted after6days of culture. Thiswas the the number of bacterial invasion.1.3Helicobacter pylori standard strains SSI invade AGS cells (MOI=10:1) tracegentamycin maintain training(25μ g/ml).Cracking cells and training on thehelicobacter pylori blood plates after infection24h,36h,48h,72h,colonies countingafter6days.1.3Observing helicobacter pylori standard strains NCTC11637infect AGS cells(MOI=100:1) under TEM. Using CLSM observe the distribution of helicobacterpylori standard strains NCTC11637in or around the AGS cells after infection.2. Using High Pure PCR Template Preparation Kit purify the genomic DNA ofhelicobacter pylori (standard strains NCTC11637and SS1, three high invasivenessclinical plants, three low invasiveness clinical plants).Analyse the genes(cagA，cag-EPIYA，cagE，babA，iceAand vacA)of helicobacter pylori by PCR.3.The influence of the cell function of AGS cells after Hp invasion.3.1Choosing0and2.5u g/ml,5μ g/ml three different concentration gradient,andusing two blockers--β1integrin antibody and Cytochalasin D block the helicobacterpylori (standard strains NCTC11637and SS1, three high invasiveness clinical plants,three low invasiveness clinical plants) invade AGS cells.3.2Previously deal with AGS cells using Cytochalasin D. Then,Cell proliferation wasdetected by CCK-8asssy and cell apoptosis was measured by TUNEL. Results1.The helicobacter pylori from different digestive disease has different invasionability.There are significant differences between them(p＜0.05).H. pylori has a higherinvasion rate with strong pathogenic ability.Proportion of intracellular H. pylori isaround10%--15%.Hp can reproduce in the AGS cells.Hp from gastric ulcer andgastric cancer have higher reproductive capacity. Helicobacter pylori can live in theAGS cell for4days.2.The genes analysis (cagA，cag-EPIYA，cagE，babA，iceAand vacA) of differenthelicobacter pyloris has no difference. There are no differences in virulence geneanalysis.3. The blockers--β1integrin antibody and Cytochalasin D all can block the Hpinvading AGS cells,and dose dependent. Cytochalasin D has better blocking effect.Previously dealing with AGS cells using Cytochalasin D, Proliferation indexesshowed an decreasing tendency in the state of low infection with H.Pylori. There aresignificant differences between them(p＜0.05).Conclusion1. The helicobacter pylori from different digestive disease has different invasionability. H. pylori can live in the cells for4days.2. H. pylori has a higher invasiveness with strong pathogenic ability and strongadhesion ability.3. There are no differences in virulence gene analysis.Invasiveness has nothing to dowith the H. pylori’s virulence genes.4. Hp invade AGS cells by β1integrin mediating and actin polymerization.The way ofblocking intracellular transduction pathway has more effective to inhibiting bacterialinvasion.5. Hp invading AGS cells can promote cell proliferation and have no impact in cellapoptosis.