| Objective: By making H.pylori culture filtrate,establish the filtrate induced gastric epithelial cells malignant transformation model.Using polygonun capitatum intervention,observe the morphological changes,detection of inflammation related indexes before and after medication of interleukin-8 and gastric cancer related factors ?-catenin?protein P53 expression level,explore the polygonun capitatum on H.pylori induced gastric epithelial cell malignant transformation,for the clinical treatment of H.pylori infection disease and provide theoretical basis for the research and development of new drug.Methods:(1)Cultivate H.pylori,preparation of filtrate after stability batches,in vitro culture comes from people immortalized gastric mucosa epithelial cell lines(Human gastric mucosa epithelial cells,GES-1)and stability batches.Different concentrations of H.pylori filtrate(6?l/mL?12?l/mL?18?l/mL?24?l/mL?30?l/mL?36?l/mL?42?l/mL?48?l/mL?54?l/mL?60?l/mL)cultivate with GES-1 cells for 14 d,fixed with methanol,Giemsa staining,counting survival cell colony,find the inhibition concentration,that half cell colony formation(IC50),select2/3IC50 concentration values for transformation test.(2)Cultivate the GES-1 cells,with different concentration polygonum capitatum(8?g/mL ? 16?g/mL ? 32?g/mL ?64?g/mL ? 128?g/mL ? 256?g/mL ? 512?g/mL ? 1024?g/mL)co-cultured for24 h,48h,72 h,used method of MTT to detect the proliferation of GES-1 cells in vitro after polygonum capitatum.(3)Choose appropriate concentration of H.pylori filtrate to co-culture with GES-1 cells,3-5 d in liquid at a time,every time in liquid or extend to join H.pylori culture filtrate,continuous culture transformation experiment for a month,after the success of the transformation to choose the right concentration of polygonum capitatum to intervene.(4)The cells were divided into control group,model group and polygonum capitatum group,the control group to do any special treatment;model group after a mon transformed GES-1 cells,polygonum capitatumgroup is the transformed cells by specific concentration of polygonum capitatum prognosis.Using inverted phase contrast microscope,transmission electron microscope to observe morphology of three groups of cells;the 10 d continuous determined by MTT method detection of three groups of cells proliferation;to extract the supernatant fluid of three groups of cells,respectively,adopt the method of ELISA detecting inflammation factor expression of IL-8 levels;extraction of three groups of cells in the cytoplasm and nucleus,detect the expression levels of ?-catenin,P53 protein using the method of Western Blotting.Results:(1)Polygonum capitatum concentration is lower than 128?g/mL,the proliferation of GES-1 cells had no obvious inhibitory effection.(2)H.pylori filtrate is lower than the concentration of the 36?l/mL,proliferation GES-1 cells had no obvious inhibitory effection.(3)After a mon co-cultured of H.pylori filtrate and GES-1,transmission electron microscopy was found GES-1 cells appeared.Cells size,and nuclear cytoplasm ratio was increased significanty,more nucleoli,heterochromatin increased,nucleus shape distortion,karyotheca depression,discontinuous?atypia,the filtrate was confirmed by H.pylori induction of malignant transformation of GES-1 cells was successfully established.(4)The cells of model group and the polygonum capitatum group are activity proliferation than the control group(P<0.05).The IL-8 in the supernatant fluid secretion of model group and the polygonum capitatum group increases significantly compared with control group(P<0.05).The expression of IL-8 was significantly lower in group of polygonum capitatum,compared with the model group(P<0.05).The expression of P53 was significantly up regulated in the group of model and the polygonum capitatum,the rate of increasion wsa more obvious,compared with the control group.(P<0.05).The expression of ?-catenin protein in model group was significantly upregulated than control group.(P<0.05).Conclusion:(1)H.pylori filtrate can induce the GES-1 cells malignant cell transformation,malignant transformation cells are atypia,the proliferation rate increased significantly.(2)Polygonum capitatum can lowregulated the expression of IL-8 and its anti-inflammatory effect is remarkable.(3)Polygonum capitatum may have not obvious effect from the regulation of P53 and ?-catenin expression to inhibitthe malignant transformation... |
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