Objective To 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).Methods Human 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 (FN 1),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.Results The 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 ofFN1 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 and 0.260 ± 0.077 in the control group,TGF-β1 group and TGF-β1 + CGP group,with significant differences among three groups (F=181.90,48.85;both at P＜0.01),and the expressions FN1 and PAI1 proteins were significantly higher in the TGF-β1 than those in the control group and TGF-β1 +CGP group (all at P＜0.05).The cell proliferative rate in the TGF-β1+CGP group was (79.30±3.44) ％ and (54.80±7.39) ％ at the third day and seventh day after culture,which were significantly reduced in comparison with (99.50 ± 1.00)％ and (99.10±0.50)％ in the control group as well as (95.10±4.20)％ and (92.10±4.50)％ in the TGF-β1 group (all at P＜0.05).The migration distance was disappeared in the TGF-β1 group,and the scratch width was not obviously changed in the TGF-β1 +CGP group.Conclusions Src inhibitor can inhibit EMT process of ARPE19 cells induced by TGF-β1,indicating that Src signaling pathway may play a critical role in EMT of RPE cells.%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3d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).细胞处理后3d,TGF-β1 +CGP处理组细胞增生率为(79.30±3.44)％,与对照组的(99.50±1.00)％和TGF-β1处理组的(95.10±4.20)％比较均明显下降,差异均有统计学意义(均P＜0.01);处理后7d,TGF-β1+CGP处理组细胞增生率为(54.80±7.39)％,明显低于TGF-β1处理组的(92.10±4.50)％和对照组的(99.10±0.50)％,差异均有统计学意义(均P=0.004).细胞划痕试验显示,TGF-β1处理组细胞迁移能力明显增强,TGF-β1+CGP处理组划痕宽度无明显变化. 结论 Src激酶抑制剂CGP能够抑制由TGF-β1诱导的ARPE19细胞EMT,提示Src信号通路与EMT过程有关.
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