| Objective1. To investigate the effect of Helicobacter pylori ATCC26695 and SS1 strains on apoptosis, proliferation, adhesion, migration of gastric epithelial cells.2. To investigate the expression of LAIR-1 and its relationship with the apoptosis of THP-1 cells induced by Helicobacter pylori ATCC26695. While provide informations to fundamental research for understanding the pathogenic mechanism of Helicobacter pylori.Methods1. We cocultured H. pylori ATCC26695 and SS1 strains with GES-1 cells for 0 h?3 h?12 h or 24 h, respectively. The control groups were cultured for 0 h?3 h?12 h or 24 h without any type of H.pylori. The change of the apoptosis in GES-1 cells was detected by the Flow cytometry. EdU assay was used to detect the cell proliferative activity. The cell adhesion of each group was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The treated cells were observed by Living Cell Working Station and scratch motility(wound-healing) assay was used to detect the cell migration velocity.2. The THP-1 cells were stimulated respectively with the H. pylori ATCC26695 for 6 h?12 h?24 h or 48 h in vitro. The apoptosis of THP-1 and the expression levels of LAIR-1 protein were assayed by flow cytometry. Three specific LAIR-1 siRNAs were transiently transfected into THP-1 cells by Lipofectamine RNAiMAX, then the changes in THP-1 mRNA and protein level were detected by semi-quantitative RT-PCR and flow cytometry, respectively. The cells transfected by the most effective siRNA were stimulated with H. pylori bacteria for 24 h. Flow cytometry was applied to examine the apoptosis of THP-1 cells.Results1. The GES-1 cells apoptosis rates were significantly increased at 3,12 and 24 h after ATCC26695 and SS1 strains infection and they in the ATCC26695 strains infected groups were all significantly higher than those in SS1 strains challenged groups.2. No significant alternation of GES-1 cells proliferative rates was observed in both ATCC26695 and SS1 strains stimulated groups during the whole experiment period.3. The treatment with ATCC26695 and SS1 strains can both induce a significant decline of GES-1 cells adhesion rates in a time-dependent manner.4. After the simulation of ATCC26695 strains,the GES-1 cells migration rates increased every hour during the whole experimental period compared to the control group. However, the GES-1 cells migration rates were almost stable and the same as the control level after SS1 strains challenge.5. ATCC26695 induced the apoptosis and decrease the expression of LAIR-1 in THP-1 cells. After THP-1 cells were infected with ATCC26695 at 4 times (6h,12 h,24 h and 48 h after treatment), cell apoptotic rates were (84± 1.77)%?(69.13±4.29)%?(48.3±3.37)% and (21.1±4.67)%, and the expression of LAIR-1 protein were 14934±178, 14369 ±244, 12259±523, 7438±539. The cell apoptotic rate and expression of LAIR-1 protein of the control cells were (4.3±2.17)%? 15576±626,respectively.6. All three LAIR-1siRNA down-regulated the expression of LAIR-1 in both mRNA and protein levels, the most effective one was siRNA2. The expressions of LAIR-1 was down-regulated, of which the mRNA inhibiting rate in 48 h was(39.1±4.67)% and the protein inhibiting rate was (38.4±3.18)%.7. After LAIR-1 expression of THP-1 cells was down-regulated for 48 hours, the cells infected with ATCC26695, apoptotic rates of LAIR-1 siRNA2 group[(36.9±3.2)% ] were significantly lower than those of negative control group[(56.1±9.8)%] (P<0.05).Conclusions1 . Different H. pylori strains had different influence on cell apoptosis,proliferation, adhesion and migration . The ability of gastric epithelial cells reconstruction and rehabilitation was different among H. pylori strains.2 . Expression of LAIR-1 can be down-regulated by H. pylori infection time-dependently,which may contribute to the inhibition of cell apoptosis induced by the bacteria. |
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