Src激酶抑制剂CGP77675对TGF-β1诱导的视网膜色素上皮细胞上皮-间质转化的抑制作用  

Inhibiting effects of Src kinase inhibitor on TGF-β1 induced epithelial-mesenchymal transition of human RPE cells

作　　者：何建锋[1] 吕立夏 罗俊杰 李宗义 沈俊慧 徐国彤 高芙蓉 

机构地区：[1]同济大学眼科研究所临床视觉科学实验室,上海200092 [2]同济大学医学院再生医学系,上海200092 [3]同济大学附属第十人民医院眼科,上海200092

出　　处：《中华实验眼科杂志》2018年第1期5-11,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：国家重点基础研究发展973计划项目（2013CB967501、2015CB964601）;上海市卫生局青年科研项目（20124y043）

摘　　要：目的探讨Src激酶抑制剂CGP77675（CGP）对转化生长因子β1（TGF-β1）诱导的人视网膜色素上皮（RPE）细胞上皮－间质转化（EMT）过程的作用及其机制。 方法将培养的人RPE细胞株（ARPE19）分为对照组、TGF-β1处理组和TGF-β1＋CGP处理组，并根据分组用相应药物处理细胞。细胞体外培养后3 d于光学显微镜下观察各组细胞的形态变化；分别采用实时荧光定量PCR法、Western blot法和免疫荧光染色法检测各组细胞中EMT相关基因及其蛋白的表达变化，包括细胞中纤溶酶原激活物抑制蛋白-1（PAI1）和纤连蛋白-1（FN1）的相对表达量及上皮细胞中闭锁小带蛋白1（ZO1）和细胞骨架蛋白F-肌动蛋白（F-actin）的分布；采用MTT法检测各组细胞的增生率；采用划痕试验检测各组细胞的迁移能力。 结果对照组ARPE19细胞呈上皮样形态，F-actin和ZO1沿细胞膜表达；TGF-β1处理组细胞为纤维样，F-actin表达的排列紊乱，细胞膜上ZO1表达不连续。TGF-β1＋CGP处理组细胞维持上皮样形态，F-actin和ZO1表达清晰且完整。对照组、TGF-β1处理组和TGF-β1＋CGP处理组细胞中FN1 mRNA和PAI1 mRNA相对表达量总体比较差异均有统计学意义（F＝33.14、82.92，均P〈0.01），对照组细胞中FN1 mRNA和PAI1 mRNA相对表达量为0.211±0.080和0.116±0.073，TGF-β1＋CGP处理组细胞中FN1 mRNA和PAI1 mRNA相对表达量分别为0.368±0.097和0.362±0.048，均明显低于TGF-β1处理组的1.000±0.001和1.000±0.001，差异均有统计学意义（均P〈0.05）。3个组细胞中FN1和PAI1蛋白相对表达量总体比较差异均有统计学意义（F＝181.90、48.85，均P〈0.01），对照组细胞中FN1和PAI1蛋白相对表达量分别为0.166±0.055和0.327±0.066，TGF-β1＋CGP处理组FN1和PAI1蛋白相对表达量分别为0.143±0.030和0.260±0.077，均明显低于TGF-β1处理组的1.000±0.001和1.000±0.001，差异均有统计学意义（均P〈0.05）�ObjectiveTo investigate the inhibiting effect of CGP77675 （CGP）, a Src inhibitor, on epithelial-mesenchymal transition （EMT） of human retinal pigment epithelial （RPE） cells induced by transformation growth factor-β1 （TGF-β1）. MethodsHuman RPE cell line （ARPE19 cells） was cultured in vitro and divided into control group, TGF-β1 group and TGF-β1＋ CGP group.Corresponding agent was added into culture medium based on grouping.The morphology of the cells were examined under the optical microscope 3 days after culture.The expressions of EMT-related genes and proteins in the cells were detected by real-time quantitative PCR and Western blot, respectively, including fibronectin 1 （FN1）, and plasminogen activation inhibitor 1 （PAI1）, and the expressions of zonula occludens protein 1 （ZO1） and cytoskeleton protein filamentous actin （F-actin） were detected by immunofluorescence staining.MTT assay was employed to evaluate the cell proliferation rate.The migration distance of the cells was measured by scratch test. ResultsThe ARPE19 cells in the control group showed an epithelial-like morphology and F-actin and ZO-1 were expressed along cell membrane.In the TGF-β1 group, the cells appeared to be fibrous-like, and the fluorescence staining of F-actin was disordered and ZO-1 was discontinuous on the cell membrane.The cells in the TGF-β1＋ CGP group remained to be an epithelial-like in shape with clear and complete expressions of F-actin and ZO-1.The relative expressions of FN1 mRNA and PAI1 mRNA in the cells were 0.211±0.080 and 0.116±0.073, 1.000±0.001 and 1.000±0.001, 0.368±0.097 and 0.362±0.048 in the control group, TGF-β1 group and TGF-β1＋ CGP groups, showing significant differences among the groups （F=33.14, 82.92; both at P〈0.01）, with the highest expressions of FN1 mRNA and PAI1 mRNA in the TGF-β1 group （all at P〈0.05）. The relative expressions of FN1 and PAI1 proteins were 0.166±0.055 and 0.327±0.066, 1.000±0.001 and 1.000±0.001, 0.143± 0.030

关 键 词：上皮-间质转化 视网膜色素上皮细胞 Src激酶抑制剂 增生性玻璃体视网膜病变 

分 类 号：R774.1[医药卫生—眼科]