| Gastric cancer is a common gastrointestinal malignant,and its incidence and mortality are increasing year after year.In China,the incidence and mortality are all the third of the total incidence and mortality of cancer.Helicobacter pylori(H.pylori)is considered the first class of carcinogenic factor of gastric cancer.The virulence factors released by bacteria and the chronic inflammation are involved in the process of malignant transformation of gastric mucosa cells.The carcinogenicity of H.pylori as single factor is not particularly strong,and many internal and external synergistic facters may enhance the carcinogenicity of H.pylori,such as the synergistic effect of nitrite.The relationship between H.pylori infection and gastric cancer,and the cellular and molecular mechanism of H.pylori induced malignant transformation of gastric epithelial cells are still unclear.To clarify the pathogenesis of H.pylori long-term chronic infection is significant to early prevention,diagnosis and treatment of gastric cancer.【Objective】To imitate the clinical infection process of H.pylori,we established the gastric mucosal epithelial cell and mice model of H.pylori chronic infection,and explored the cellular and molecular mechanism of malignant transformation of mucosal epithelial cells induced by H.pylori alone or in combination with MNNG chronic infection.【Mehtods】1 In this study,we used immortalized human gastric epithelial cells line GES-1 as the target cell,CagA~+H.pylori J99 alone or in combination with MNNG was used to infect GES-1 cells for 1 year.The cell morphology was observed by light and electron microscope;cell proliferation and apoptosis were detected by MTT colorimetric assay,clony-forming experiment and Annexin V/PI double staining method;the migration and invasion ability of cells were investigated by transwell and wound‐healing assay;ELISA was used to detect the concentration of IL-8;sphere‐formation assay and cell surface markers were used to detect CSC-like and EMT-like changes of cells;RT-PCR,Western blotting and immunofluorescence staining were used to detect the mRNA and protein expression of related genes.2 Taking C57BL/6J mice for research objects,CagA~+H.pylori SS1 stain was administrated to the stomach alone or in combination with MNNG to infect the gastric mucosa repeatedly for one year.The animal model of H.pylori chronic infection was established,and some mice were killed regularly.The serum and gastric mucosa tissue samples were collected.The infection rate of H.pylori was detected by the rapid urease test,warthin starry silver staining;the concentration of IL-8 in serum was detected by ELISA;the histomorphology of gastric mucosa was observed by HE-staining under a light-microscrope and ultrastructures under an electron microscope;Western blotting and immumohistochemistry were used to detect the expression of related proteins of cell proliferation,apoptosis,EMT and CSC.3 72 samples of gastric cancer were collected,which were diagnosed by clinicopathology and resected by operation,and the gastric cancer and its adjacent tissues were obtained.The gastric cancer tissues were divided into H.pylori infection group(40 cases)and H.pylori non infection group(32 cases)by rapid urease method and warthin starry silver staining.The histomorphology of gastric mucosa was observed by HE staining.The expression of IL-8 and Wnt2 in gastric cancer and its adjacent tissues were detected by immunohistochemistry.The relationship between H.pylori infection rate,IL-8,Wnt2 expressions and pathological and clinical data was analysed by statistics.And the relationship between H.pylori infection,IL-8 expression,Wnt2expression and prognosis of gastric cancer patients was analyzed by survival curve and survival regression.【Results】1 After long-term chronic infection with CagA~+H.pylori,with the prolongation of infection time,gastric mucosa GES-1 cells occurred abnormal proliferation,inhibition of apoptosis and drug resistance,increased expression and secretion of IL-8,enhanced migration and invasion ability,and significantly increased the expressions of CSC-like and mesenchymal cell-like markers.Meanwhile,NF-κB and Wnt/β-catenin signaling pathway all were activated.MNNG can significantly enhance the inductive effect of H.pylori chronic infection on GES-1 cells.It was suggested that long-term chronic infection of CagA~+H.pylori can induce CSC-like and mesenchymal cell-like changes in gastric epithelial cells under the joint regulation of NF-κB and Wnt/β-catenin pathway.2 CagA~+H.pylori SS1 strain can successfully infect the gastric mucosa of mice.Under the stimulation of long–term,recurrent,chronic H.pylori infection,in the gastric mucosa of mice,pathological changes such as abnormal hyperplasia,mucous cysts,adenomatoid polyposis and precancerous lesions gradually occurred,the expression and release of IL-8 increased and the inflammatory reaction occurred evidently.The expressions of Cyclind D1,Bcl2 and Bax proteins showed abnormal hyperplasia and reduced apoptosis of gastric mucosa cells.The markers of CSC-like and mesenchymal cell-like in mouse gastric epithetlial cells increased like GES-1 cell,and the NF-κB and Wnt/β-catenin signal pathways were activated abnormally.The infection effect of H.pylori was significantly improved by MNNG.It was suggested that the Wnt/β-catenin/IL-8 pathway in gastric mucosa of mice was activated abnormaly by CagA~+H.pylori long-term chronic infection,and the inflammatory reaction,CSC-like,EMT-like and precancerous pathological changes of gasric mucosa cells were observed.3 The expression of IL-8 and Wnt2 in gastric cancer was significantly higher than that in normal mucosa.The expression of IL-8 and Wnt2 in gastric cancer with positive infection of H.pylori was significantly higher than that of uninfected gastric cancer,and the expression of IL-8 was positively correlated with Wnt2.The positive rate of IL-8 and Wnt2 in the early stage of gastric cancer without lymph node metastasis was significantly lower than those in the advanced stage with lymph node metastasis;the survival rate of gastric cancer patients with positive expression of IL-8 and Wnt2was significantly lower than those with negative expression.【Conclusions】CagA~+H.pylori chronic infection and MNNG activate Wnt/β-catenin/NF-κB/IL-8 regulatory pathway of gastric mucosal cells,induce abnormal proliferation of gastric mucosal epithelial cells and CSCs-like and mesenchymal cell-like changes,promote inflammatory response of gastric mucosa and precancerous transformation.In clinic,the activation of Wnt/β-catenin and NF-κB pathways in H.pylori positive gastric cancer decreases the survival of the cases. |
PDF Full Text Request