MiRNA-29b Regulates P53 Expression In Tenon's Capsule Fibroblasts Of Normal Human Subconjunctival And Its Significance

Objective To study the process of scar formation in the filtration tract after glaucoma surgery.Culture primary human Tenons' fibroblasts in vitro and detect the expression relationship between the level of micro RNA-29 B and p53 in the normal fibroblasts and the activated myofibroblasts.To investigate the mechanism of filtration scar formation after glaucoma filtration surgery.Methods Get the normal Tenon's tissue from the patients of ophthalmological strabismus surgery in The first Affiliated Hospital of Anhui Medical University ophthalmic operating room from March 2016 to July 2016,cultured primary fibroblasts by tissue culture method,passage to 2-4 generations for experiment.Activated Fibroblasts into myofibroblasts by TGF-?1(10ng / ml).Detected the expression of p53 m RNA and protein in fibroblasts and myofibroblasts by q PCR and IHC respectively.Transfect p53 with si RNA to inhibit the expression of in fibroblasts,then use q PCR to detect the expression of mi RNA-29 b and p53 in fibroblasts,activated myofibroblasts and transfected myofibroblasts.Then use SPSS16.0 software and other software to analysis the data.Results 1.About 2 weeks later cells crawling out from the edge of the organization,6-8 weeks later cells were covered on cell culture flask bottom,fibroblasts are visible under 10 times the magnification of the inverted microscope with large volume,a typical flat fusiform.The nucleus of fibroblasts with a regular oval is in the center,cells between each other arranged in a spiral.Cells were passaged and then activated with TGF-?1(10ng / ml,48h).The cells were activated by fibroblasts into myofibroblasts.The cell volume increased and the cells were irregular in shape with frizzled edges with disordered arranged.2.After digestion of fibroblasts and activated myofibroblasts, respectively.Follow the immunohistochemical steps,after DAB staining,the nucleus was stained deeply under the inverted microscope,the cytoplasm is not colored,which means P53 protein is expressed in the nucleus,diffuse distribution,no expression in the cytoplasm.According to the color depth,the expression of p53 protein in MFs was significantly higher than that in HTFs group.According to the degree of colour of pictures,use image proplus software to analyse all the immunohistochemical pictures,Independent sample mean t-test showed that: the data of each group obey the normal distribution,The expression of p53 protein in MFs group(0.0419 ± 0.0124)was higher than that in HTFs group(0.0089 ± 0.0085)in both groups,t=-10.384,P<0.05,N=3,The difference has statistically significant.3.Extract m RNA from HTFs and MFs cells,then reverse transcript m RNA to c DNA,finally compare relative expression of p53 m RNA by q PCR.Use Independent samples t-test to analyze the expression of p53 in HTFs and MFs.The results shows that compared with HTFs(1 ± 0.0672),the expression of p53 in MFs(4.8650 ± 0.2577)was significantly increased,t =-19.821,p <0.05,N = 3,the difference was statistically significant.Extracted m RNA from the HTFs cells,MFs cells and si RNA transfected cells,then reverse transcript m RNA to c DNA,the relative expression of p53 m RNA was verified by q PCR to see if p53 was transfected successfully.Then relative expression of mi RNA-29 b was detected by q PCR.q PCR results showed that the expression of mi R-29 b in three groups of cells from high to low were: si RNA-p53 transfected group,normal HTFs group,MFs activated group,The Kruskal-Wallis H test was used to analyze the expression of p53 and mi R-29 b in each group of cells.In each group,p53: HTFs group 1(1,1),MFs group 3.9750(1.6350,6.2452),si RNA transfection group 0.0560(0.0112,0.1195)and negative control group 0.9750(0.8300,1.1200).mi R-29b: 1(1,1)in HTFs group,0.5450(0.4500,0.6100)in MFs group,5.0500(3.0875,4.1700)in si RNA transfection group and 1.0250(0.9475,1.1250)in negative control group.Using the Kruskal-Wallis H test analysis the statistical of the p53 m RNA data of each group,X2 = 15.928,with aP value of 0.000 on both sides,suggesting differences between the groups,All the groups were compared with each other,except the negative control group and HTFs group P> 0.05,the difference was not statistically significant,the other groups in the expression of p53 were different,p <0.05,the difference was statistically significant.Similarly,mi R-29 b data of each group were analyzed by Kruskal-Wallis H test,X2 = 22,bilateral P values of 0.000,differences between the groups.All the groups were compared with each other,except the negative control group and HTFs group P> 0.05,the difference was not statistically significant,the rest of the group in the expression of mi R-29 b were different,p <0.05,the difference was statistically significance.5.Comparing the migration ability of HTFs cells and MFs cells by scratch test,it was found that after the activation of HTFs to MFs,the migration ability of cells increased.Conclusion In MFs cells,the expression of p53 m RNA and p53 protein are increased,while the expression of mi RNA-29 b is decreased.After the expression of p53 downregulated,the expression of Mi RNA-29 b is up-regulated.,which indicated that Mi RNA-29 b may inhibit the formation of filtration scar in glaucoma by down-regulating the expression of p53.