| BackgroundAt present,Gstric carcinoma is one of the common clinical digestive tract cancer,stomach cancer accounted for about 95% of malignant tumors of the digestive tract,。In malignant diseases diagnosed each year,stomach cancer ranked fourth,is the world after the incidence of lung cancer mortality,ranked second in the world,has seriously affected the people’s health health.The discovery of human target sites is a new breakthrough in cancer therapy.It brings hope for cancer patients.It mainly plays the role of inhibiting tumor cells and promoting tumor cell apoptosis by combining targeted drugs with specific targeting sites of human body.At present,there are many target loci for the treatment of gastric cancer,but most of them are not mature.Studies have shown that FHL2(Four and a half LIM domain 2)is closely related to the occurrence,development and prognosis of gastric cancer.By studying the composition,function and transmission pathway of FHL2,it may provide a new target site for tumor targeted therapy.Although FHL-2 is one of the most popular target sites at present,there are many questions in the study of FHL2,so it can not be used in clinical application.In this paper,we studied the expression and significance of FHL2 protein and its related proteins in gastric cancer,and further understand the relationship between FHL2 and gastric cancer,and provide a new target for targeted therapy of gastric cancer.Purpose1.the establishment of epithelial mesenchymal transition model of gastric cancer cells;2.reveal the correlation between FHL-2 and epithelial mesenchymal transition in gastric cancerMethod1.Establishment of epithelial mesenchymal transformation model of gastric cancer cells1.1 Induction of interstitial like phenotype of gastric epithelial cells in gastric cancerInterstitial like test group: 10ug/ml oxaliplatin was used to stimulate gastric cancer cell line SGC-823(epithelial phenotype)and induce it to become epithelial mesenchymal like phenotype of gastric cancer.Control group: normal human SGC-823(epithelial phenotype).1.2 Morphological observationThe culture fluid of the culture bottle was poured down,and the cells were washed with 1-2mlPBS solution for 2 times.The residual PBS solution was carefully removed by pipette,and 1640 5ml containing 10% fetal bovine serum was added.The control group and the experimental group were observed by inverted phase contrast microscope.First,the cell morphology was observed from the 100 X microscope,and two typical cells were observed.Then,200 X cell microscope was used to observe and record the morphological changes of cells.1.3 Transwell experimentThe experiment of a gastric cancer cell line SGC-823 in logarithmic growth(epithelial cell phenotype)and induce a mesenchymal cell phenotype,placed in the chamber on the chamber,adjust the concentration of the cell suspension of 2.5 * 105 /ml,500 L 10% 24 h were cultured with the medium containing fetal bovine serum and down in the room,with a cotton swab to scrape by without membrane cells in the room,room temperature,were fixed and stained with PBS solution,then quickly washing,drying,and then change the inverted microscope to observe two groups of cell invasiveness.Evaluation of epithelial mesenchymal transition model of gastric cancer cells.2.Expression of FHL-2,E-calcium mucin and vimentin in the epithelial mesenchymal transition model of gastric cancer cellsWestern FHL-2,E-blot experiments of E-cadherin and vimentin in gastric cancer cell epithelial mesenchymal transition expression model was used for semi quantitative analysis: different concentration of separation gel and concentrated by electrophoresis,were transferred to PVDF membrane membrane after electrophoresis,followed by staining,observe protein bands,wash well the protein bands at 4 ℃for 24 hours overnight,with TBST low shaking repeatedly washed membranes,5 minutes each time,adding two anti37 ℃hybridization after 2 hours,finally adding fluorescent agent,in darkroom exposure imaging.3.Data analysisFrom the experimental group and the control group data,using SPSS13 software,the normal distribution test,normal distribution data using the mean and standard deviation of the method that using two independent sample t test statistical analysis to test the level of α =0.05,if P > α,showed no statistically significant difference between the two groups;if P < α,showed statistically significant difference between the two groups.Result1.Establishment of epithelial mesenchymal transformation model of gastric cancer cells1.1 Changes in the two groups of gastric cancer cells: morphological phenotype of the cells in the experimental group composed of epithelioid cell phenotype to mesenchymal cell phenotype change,mainly as follows: the cell size,there are obvious pseudopodia cells in the control group;the phenotype of epithelioid cell phenotype,mainly as follows: round,cell gap is small,the shape and size of more rule.1.2 Transwell experimental result:The number of cells passing through the membrane in the control group was 65.7±11.65,and the number of cells passing through the membrane in the experimental group was 123.4±28.13.The calculated T=12.434,P< 0.05,the difference was statistically significant,and the invasive ability of the experimental group was significantly higher than that of the control group.2.Expression of HL-2,E-calcium mucin and vimentin in the epithelial mesenchymal transition model of gastric cancer cellsWestern bolt results: compared with the control group,the expression level of vimentin and FHL2 in the experimental group was increased,while the expression of Ecadherin was down regulated,the difference was statistically significant.Conclusion1.The sustained stimulation of oxaliplatin is an effective method for the establishment of epithelial mesenchymal transition model of gastric cancer cells.2.FHL-2 protein and gastric cancer cell epithelial mesenchymal transition process is closely related to FHL-2 may become a key site of tumor targeted therapy,in epithelial mesenchymal transition occurs in gastric cancer cells,the expression of intervention of FHL2 may provide a new idea for reversing the process. |
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