正常人眼结膜成纤维细胞转化为肌成纤维细胞过程中p53的表达变化及其对微小RNA-29b表达的影响  

Expression of p53 and its effect on microRNA-29b expression in the process of normal human conjunctival fibroblasts transforming to myofibroblast

作　　者：晏维玲 廖荣丰[1] 李宁[1] Yan Weiling;Liao Rongfeng;Li Ning(Department of Ophthalmology,the First Affiliated Hospital of Anhui Medical University,Hefei 230031,China)

机构地区：[1]安徽医科大学第一附属医院眼科,合肥230031

出　　处：《中华实验眼科杂志》2018年第9期676-681,共6页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金青年基金项目(81500716)

摘　　要：目的检测正常人Tenon囊成纤维细胞(HTFs)活化为肌成纤维细胞(MFs)过程中微小RNA(miR)-29b和p53的表达水平,探讨p53表达变化对miR-29b表达的影响。方法收集安徽医科大学第一附属医院2016年3—7月行眼科斜视手术术眼正常Tenon囊组织,用组织块培养法培养出原代HTFs,传代至2~4代并进行实验。将细胞分为4个组:HTFs组细胞常规培养;TGF-β1活化组细胞用10 ng/ml TGF-β1处理;将成纤维细胞活化为肌成纤维细胞;siRNA转染组及阴性对照组分别采用含p53 siRNA以及阴性siRNA转染细胞后经10 ng/ml TGF-β1活化。分别采用免疫组织化学染色法和细胞划痕实验检测HTFs组和TGF-β1活化组细胞p53蛋白表达水平及细胞迁移能力。采用荧光定量PCR法检测各组细胞miR-29b和p53 mRNA相对表达量。结果原代HTFs呈长梭形,细胞间呈螺旋状排列,TGF-β1活化后细胞转化为MFs,细胞形态不规则,排列呈无序状态。TGF-β1活化组细胞中p53蛋白染色强度显著强于HTFs组,差异有统计学意义(t=-10.384,P<0.05)。TGF-β1活化组细胞的相对迁移距离明显大于HTFs组细胞,差异有统计学意义(t=9.750,P=0.000)。HTFs组、TGF-β1活化组、siRNA转染组及阴性对照组细胞中p53 mRNA相对表达量分别为1.00±0.00、3.95±2.61、0.06±0.06和0.98±0.16,miR-29b相对表达量分别为1.00±0.00、0.54±0.09、5.10±2.31和1.03±0.09,总体比较差异均有统计学意义(F=6.688,P=0.006;F=13.640,P=0.000)。TGFβ1活化组细胞p53 mRNA相对表达量显著高于HTFs组和siRNA转染组,miR-29b相对表达量显著低于HTFs组和siRNA转染组,差异均有统计学意义(均P<0.05)。结论在HTFs转变为MFs过程中,p53显著升高,miR-29b明显降低;抑制p53表达后,miR-29b表达升高。p53与miR-29b之间可能存在相互作用来调控纤维化瘢痕的形成。Objective To investigate the relationship between p53 and miR-29b in myofibroblasts(MFs)induced from human Tenon fibroblasts(HTFs).Methods Human Tenon tissues were obtained from strabismus surgery patients in the First Affiliated Hospital of Anhui Medical University from March to July 2016.The primary HTFs were cultured by tissue culture method and passaged to 2-4 generations.The cells were divided into 4 groups:HTFs group cells were normally cultured,the TGF-β1 activation group cells was activated with 10 ng/ml tumor necrosis factorβ1(TGF-β1),the siRNA transfection group and the negative control group were transfected with p53 siRNA and negative siRNA,respectively.The expression of p53 protein and cell migration ability of HTFs and MFs were detected by immunohistochemical and cell scratch assay.The relative expression level of miR-29b and p53 mRNA in each group was detected by real-time PCR.This study followed the Helsinki declaration and the patients or their guardians signed informed consent.Results The primary HTFs had long fusiform shape,and were arranged in a spiral shape.After activated by TGF-β1,the cells were transformed into MFs,and the cell morphology was irregular and the arrangement was disordered.The staining intensity of p53 protein in TGF-β1 activation group was significantly stronger than that in HTFs group(t=-10.384,P<0.05).The migration distance of TGF-β1 activation group cells was significantly larger than that of HTFs group(P<0.05).The relative expression level of p53 mRNA was 1.00±0.00,3.95±2.61,0.06±0.06 and 0.98±0.16 in HTFs group,TGF-β1 activation group,siRNA transfection group and negative control group,respectively;the relative expression levels of miR-29b was 1.00±0.00,0.54±0.09,5.10±2.31 and 1.03±0.09 in HTFs group,TGF-β1 activation group,siRNA transfection group and negative control group,respectively;the overall differences were statistically significant(p53 mRNA:F=6.688,P=0.006;miR-29b:F=13.640,P=0.000).The relative expression of p53 mRNA was significantly higher an

关 键 词：青光眼 瘢痕 P53 微小RNA-29b 

分 类 号：R779.6[医药卫生—眼科]