结缔组织生长因子对人Tenon囊成纤维细胞中E-cadherin蛋白表达的促进作用

Promoting effect of connective tissue growth factor on expression of E-cadherin in human Tenon capsule fibroblasts

背景 青光眼滤过手术后滤过通道的瘢痕化是手术失败的主要原因,其主要病理机制是成纤维细胞的异常增生、间质-上皮转分化及细胞外基质的重塑.结缔组织生长因子（CTGF）是促进瘢痕形成的关键因子,而CTGF是否能促进人Tenon囊成纤维细胞（HTFs）的间质-上皮转分化尚不清楚.目的 观察CTGF对HTFs间质-上皮转分化的影响.方法 用体积分数10％胎牛血清的DMEM高糖型培养液进行HTFs体外常规培养和传代,取3～6代细胞用于实验.将培养的HTFs分为空白对照组和CTGF处理组,空白对照组用DMEM完全培养液培养细胞,CTGF处理组在培养液中加入CTGF,使终质量浓度为50 ng/ml.细胞培养后48 h,采用细胞免疫荧光染色技术鉴定HTFs中上皮钙黏素（E-cadherin）蛋白的表达,采用Western blot法对HTFs中E-cadherin蛋白的表达量进行检测.结果 空白对照组及CTGF处理组HTFs均生长良好,呈长梭形,漩涡状排列.细胞免疫荧光染色显示,CTGF处理组HTFs细胞质呈红色荧光,细胞核呈蓝色荧光;空白对照组HTFs仅见DAPI蓝染的细胞核,无E-cadherin表达的红色荧光.Western blot法检测结果显示,空白对照组HTFs中E-cadherin蛋白无表达,而CTGF处理组HTFs中E-cadherin蛋白的相对表达量为0.63±0.08.结论 间叶组织来源的成纤维细胞本身不表达E-cadherin,在CTGF的刺激下成纤维细胞能够表达E-cadherin,CTGF促进HTFs的间质-上皮转分化.

Background Scarring of filtration channel following glaucoma filtering surgery is a main cause of the failure of the surgery.The proliferation,epithelial-mesenchymal transition and extracellular matrix remodeling of fibroblasts are thought to be the primary pathological mechanism of scarring.Connective tissue growth factor （CTGF） plays a promoting role in the formation of scar.Whether CTGF participates in mesenchymal-epithelial transition of human Tenon capsule fibroblasts （HTFs） is not clear yet.Objective This study attempted to investigate the effect of CTGF on the mesenchymal-epithelial transition of HTFs in vitro.Methods HTFs were cultured and passaged in high glucose DMEM medium with 10% fetal bovine serum,and the cells of generation 3-6 were used in this experiment.The cells were divided into the blank control group and CTGF-treated group and were routinely cultured in the blank control group.CTGF was added in the medium in the CTGF-treated group,with the final concentration 50 ng/ml CTGF.Immunofluorescence staining was used to identify and locate the expression of E-cadherin protein in the cells,and Western blot assay was employed to quantitatively analyze the expression level of E-cadherin protein in 48 hours after culture.Results The HTFs grew well with the spindle-like shape and vortex-like arrangement.The red fluorescence （E-cadherin protein） in the cytoplasm and blue fluorescence in the cellular nucleus were seen in the CTGF-treated group,but only nucleus with blue fluorescence were obtained in the blank control group.Western blot assay showed that the E-cadherin protein expression was absent in the blank control group,however,the relative expression level of E-cadherin protein in the cells was 0.63± 0.08.Conclusions E-cadherin protein is not expressed in fibroblast derived from mesenchymal tissue.However,CTGF can induce the expression of E-cadherin in HTFs.This study suggests that CTGF promotes the mesenchymal-epithelial transition of HTFs in vitro.