Effect Of H.pylori On Pepsinogen Expression In Primary Human Gastric Epithelial Cells

BackgroundHelicobacter pylori is a major cause of chronic gastritis and peptic ulcer and is closely related with the occurrence of gastric cancer.But the carcinogenic mechanism by H.pylori hasn't yet been fully explained.Human pepsinogen(PG)is the inactive precursor of specific functional enzyme-pepsin of the gastric mucosa,which is mainly produced by chief cells in fundic gland,which is divided into pensinogen A and pepsinogen C,with different biochemical and immunological properties.99% pepsinogen is secreted into the stomach lumen and 1%is secreted into blood circulation. Serum PG levels can reflect morphology and function of different parts in gastric mucosa.At present,the relationship between H.pylori and serum pepsinogen mainly came from studies in vivo,which may be influenced by the colonization of H.pylori, the distribution of different PG subtypes cells,immune response,as well as more complex regulation systems in the body.Then,how about the effect of H.pylori on pepsinogen expression in human primary gastric epithelial cells in vitro is not reported in the literatures.An about 100bp insertion-deletion RFLP polymorphism was observed between exon 7 and 8 with several restriction enzyme.Studies reported that PGC polymorphism was associated with susceptibility to gastric ulcer and gastric cancer.Our previous study reported that PGC gene polymorphism and H.pylori infection seemed to present a positive interaction in the development of gastric ulcer and gastric cancer.But the pathogenic mechanism by H.pylori and PGC gene polymorphism hasn't yet been fully explained.Then how about the effect of H.pylori on pepsinogen expression in primary gastric epithelial cells with PGC gene polymorphism is still not clear.Immortalized cell lines are always applicated in experiments in vitro,because of its accessible acquisition,simple culture,and unlimited passage.But for a long time's cultivation the biological characteristics of the cell lines have been changed largely, which is always a puzzled problem for cell lines as experimental model.Primary culture cells make up for the shortage of immortalized cell lines because they are rapidly isolated from the body,no biological changes,still keep diploid genetic properties and most close to characteristics in vivo.Primary culture of gastric epithelial cells is developed in the past three decades,especially eighties and nineties in the last century as a result of needs of basic research and clinical treatment and the development of cell culture technology,and its source involves mice,rabbits,dogs, pigs,people and many other species.People find that function of primary gastric epithelial cells have many similarities as that of gastric epithelial cells in vivo after evaluation.H.pylori is the injury factor of gastric mucosa and the initiating factor in gastric mucosal lesion.To investigate the mechanism of H.pylori and gastric diseases in cellular level,H.pylori-induced injury model similar to mucosal injury in vivo is the prerequisite,but by now the ideal experimental model is still not gained.The aim of this study is observe the damage of H.pylori to primary gastric epithelial cells,to establish the injure model of gastric epithelial cells induced by H.pylori and to investigate the effect of H.pylori on pepsinogen expression in primary gastric epithelial cells and gastric epithelial cells with different PGC gene alleles.The research may provide experimental basis for the mechanism of cell injury induced by H.pylori and anti-injury produced by the body.ObjectiveTo establish the injure model of primary human gastric epithelial cells induced by H.pylori.To investigate the effect of H.pylori on pepsinogen expression in primary gastric epithelial cells.To identify the effect of H.pylori on pepsinogen expression in primary gastric epithelial cells with PGC gene polymorphism.MethodsH.pylori was cultured in Brain heart infusion agar medium;Gastric epithelial cells were cultured by primary cell technique;After co-culture of H.pylori and cells, morphological changes of cell injury were observed by inverted microscope;Lactic dehydrogenase(LDH)activity was measured by LDH Kit;Sequence specific primers-Polymerase chain reaction(PCR-SSPs)were performed to analyze the genotype of PGC polymorphism of 100bp insertion-deletion fragments between 7-8 exon;Enzyme-linked immunosorbent assay(ELISA)was used to detect PGC expression in culture supernatant of gastric epithelial cells infected with H.pylori.Result1.The establishment of the injure model of primary human gastric epithelial cells induced by H.pylori①Primary culture of normal gastric epithelial cellsOn microscopic examination within the initial 24 hours of culture,gastric epithelial cells were cluster-like growth.The more the dense clusters,the faster the cell proliferation and the more the number of cells.After 2 days of culture,with the cluster as the center,cells rapidly grew to outward and connected with the cells around into a piece.After 3 days of culture,when spreaded 70%of cell plate,cells were in good condition with single-adherent,diamond-shaped or polygonal and the nuclear was large and oval.②The observation of the injury of gastric epithelial cells induced by H.pyloriAfter co-culture of H.pylori and gastric epithelial cells(100:1)for 6 hours,the injury could be observed:cell gap expanding,cell connection relaxation,cells outline blur,the debris increasing;the cell membrane and nuclear membrane became rough; occasionally vacuola degeneration observed;cell morphology became changed.LDH activity in 6 hour showed much higher than that of 3 hour and the control group,while roughly as same as that of 9 hour.2.The effect of H.pylori on pepsinogen expression in primary gastric epithelial cell.PGA protein expression in cells infected by H.pylori was higher(247.0±104.5) than that without infection(239.7±107.8,P=0.90).PGC protein expression in cells infected by H.pylori was higher(43.6±53.3)than that without infection(31.2±23.6,P =0.56).There were no statistical differences between before and after H.pylori infection.3.The effect of H.pylori on pepsinogen expression in primary gastric epithelial cell with PGC gene polymorphism. Compared with other alleles,in allele 310 of PGC gene,the change rate of PGA protein expression was higher before and after H.pylori infection.It was the same with the change rate of PGC protein in allele 310 of PGC gene compared with other alleles. There were no statistical differences among different alleles after H.pylori infection.Conclusion1.The injury model induced by H.pylori in human primary gastric epithelial cells was successfully established.2.PGA and PGC protein expressions in primary gastric epithelial cells were increased by H.pylori.3.There were different effects on PG expressions of different PGC gene polymorphism by H.pylori infection.Compared with other alleles,in allele 310 of PGC gene,the change rate of PG protein expression was higher before and after H.pylori infection.