The Research Of Oxidative DNA Damage Of Gastric Epithelial Cell Line Induced By Helicobacter Pylori

Objective Oxidative DNA damage played an important role in the pathogenesis of many kind of gastric mucosal diseases such as gastric cancer. Helicobacter pylori infection perhaps induced the significant increase of the exogenous oxidative DNA damage in gastric mucosa. However, the current research works were limited to use the method of Immunohistochemistry for testing 8-OH-dG which is the sensitive marker of the oxidative DNA damage in biopsy samples, and these data could not reflect the direct influence that helicobacter pylori did to the gastric epithelial cells. In this study, We made the supernatants of helicobacter pylori NCTC11637(CagA+, VacA+) and G50(CagA-, VacA-) coincubate with the normal gastric epithelial cell line GES-1, and then the content of 8-OH-dG in the cells was measured with Immunocytochemistry. It was aimed to discuss the potential role of the oxidative DNA damage induced by helicobacter pylori in Hp-associated diseases. Method Helicobacter pylori CagA+,VacA+ strain NCTC11637 or CagA-,VacA-strain G50 was cultured with shaking in H.pylori medium until to an optical density at 600 nm of 0.7. The supernatants were removed after centrifugation and stored at 70°C. Cell Vacuolation Assay was used to detect the presence of vacuolating toxin in the supernatants obtained from Hp. Tetrazolium Salt(MTT)Assay was used to reflect the cytotoxicity of the supernatants from Helicobacter pylori to GES-1 cells. The human gastric epithelial cell line GES-1 was treated with various concentrations of the supernatants from NCTC11637 or G50 and Hp medium without inoculating Hp strain as a control , and the coverglasses were taken out at 24,48 or 72h, and then fixed by 4% formamint. Immunocytochemistry was used to detect the content of the 8-OH-dG which is a sensitive marker of oxidative DNA damage . Results were observed under a light microscope. Result GES-1 cells were treated for 48h with 10,8,6,4 and 2mg of the supernatants/ml obtained from Hp CagA+, VacA+ strain NCTC11637 and Hp CagA-, VacA-train G50. In GES-1 cells treated with 10,8,6,4 mg/ml NCTC11637 supernatant, more than 50% of all cells showed cellular vacuoles. In contrast, the supernatant from G50 failed to induce the formation of vacuoles. Human gastric epithelial cells GES-1 were treated with various concentrations (8,6,4 and 2mg/ml) of the Hp supernatants for different time(24,48 and 72 hours)and the content of 8-OH-dG in the cells was measured with immunocytochemistry. The percentage of 8-OH-dG positive GES-1 cells was both increased with the increased concentrations of the supernatants from NCTC11637 and G50. Meanwhile, the percentage of 8-OH-dG positive GES-1 cells was both increased with the increased time for which GES-1 was treated with the supernatants from NCTC11637 and G50. There was no significant difference between the percentage of 8-OH-dG positive GES-1 cells treated with the same concentration from NCTC11637 and G50 for the same time.Human gastric epithelial cells GES-1 were treated with various concentrations of the Hp supernatants and the cell proliferation was determined by MTT Assay. Relatively high concentrations(10,8 and 6mg/ml) of the supernatants from NCTC11637 and G50significantly inhibited the proliferation of GES-1 cells, P<0.05. Treatment of the GES-1 cells with the 10mg/8mg supernatant/ml from NCTC11637 for 72h resulted in a significant decrease in the proliferation of GES-1 cells compared with 10mg/8 mg supernatant/ml from G50, and there was no significance between the proliferation of GES-1 treated with 6,4 and 2mg supernatant/ml from NCTC11637 and G50. Conclusions: 1. In vitro, the supernatants of Helicobacter pylori strain NCTC11637 and G50 both could induce the significant increases of the oxidative DNA damage in the normal gastric epithelial cells line GES-1, and this kind of damage was increased with the increased concentration of the supernatant or the increased time for which GES-1 was treated at certain degree by the way independent of CagA,VacA status. . 2. In vitro, the supernatants from NCTC11637 and G50 both could result in significant decrease in the viability of normal gastric epithelial cell line GES-1, even apoptotic morphology.