Establishment And Application Of A Novel Human Primary Gastric Epithelial Cell Model Infected With Helicobacter Pylori

ObjectivesTo cultivate 3D human gastric epithelial organoids,and further transfer into 2D primary gastric epithelial cells,which could be used as infection model for Helicobacter pylori(H.pylori).The model was then used to study the molecular mechanism of Helicobacter pylori infection in gastric epithelial cells.Methods1.Gastric glands were isolated from normal gastric epithelial tissues,and were further grown into garstric organoids in the martrigel with the medium containing various growth factors and apoptosis inhibitors.2.Characterizations and related molecular markers of gastric epithelial organoids were identified by HE staining and immunofluorescence techniques.3.The morphological changes of normal primary gastric epithelial cells infected by H.pylori were studied,and the expression levels of Cag A~+H.pylori P12 standard strain infective proteins were identified by Western blot.4.Three cases of normal gastric antrum mucosa were selected as the source of organoids.The 3D organoids were transformed into 2D primary gastric epithelial cells and infected with Cag A~+H.pylori P12 standard strain.Samples of infected and uninfected cells were collected and total RNA was extracted for RNA sequencing(RNA-seq).5.The relationship between LRP8 and H.pylori infection and gastric cancer was analyzed using TCGA and GEO database.Functional analysis of genes co-expressed with LRP8 in gastric cancer was performed using the DAVID platform.K-M Plotter database was used to analyze the correlation between high and low expression of LRP8 and prognosis of gastric cancer patients.6.The expression of LRP8 in gastric cancer cell line AGS infected with Helicobacter pylori was determined by Western blot.The expression of LRP8 in gastric cancer cell lines was determined by real-time fluorescence quantitative PCR.Results1.The gastric epithelial 3D organoids of Chinese that can be passed down for a long time have been successfully cultivated,with typical molecular markers of human gastric mucosa epithelium.In addition,the primary gastric epithelial cells from organoids cultured in 2D plane can be used as the primary cell infection model of H.pylori in vitro.2.RNA-seq results showed that the expression of LRP8 was significantly decreased in primary gastric epithelial cells infected with Cag A~+H.pylori P12standard strain(P<0.05).3.Data from GSE25146 showed that LRP8 was up-regulated in gastric cancer cell line AGS after Helicobacter pylori infection(P<0.05).In TCGA and GEO databases,LRP8 overexpression was closely associated with gastric cancer(P<0.05).Survival analysis of the two microarrays in the K-M Plotter database showed poor prognosis in patients with high LRP8 expression(P<0.05).4.Verification experiments show that,the expression of LRP8 in AGS of gastric cancer cell lines after infection increased significantly with the extension of infection time(P<0.05).Compared with normal gastric epithelial cell line GES-1,LRP8expression was significantly increased in AGS,MKN45,and NCI-N87(P<0.05).Conclusion1.We have successfully constructed a 3D gastric epithelial organoids model of Chinese people,which can be used as a persistent source of 2D primary gastric epithelial cells and bring a new individualized model for the study of the molecular mechanism of H.pylori infection of human gastric epithelial cells.2.LRP8 is significantly correlated with H.pylori infection in the stomach and gastric cancer,which may be involved in the pathogenesis of gastric cancer.