转化生长因子β_2对人视网膜色素上皮细胞间质转化的影响

The effect of transforming growth factor-β_2 on the epithelial mesenchymal transition of human retinal pigment cells

目的研究转化生长因子(TGF)-β_2对人视网膜色素上皮细胞间质转化的影响。方法体外培养的ARPE-19细胞,经不同浓度的TGF-β_2(0、1、5、10ng/mL)处理24h后,免疫荧光染色观察细胞形态学改变;Transwell小室观察细胞迁移能力的变化;实时荧光定量聚合酶链反应(RT-PCR)及蛋白质印迹法(Western blotting)方法检测上皮细胞标记物E-cad及间质细胞标记物α-平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(FN)及Ⅰ型胶原蛋白(Col-Ⅰ)的基因及蛋白表达量。结果随着TGF-β_2浓度的增加,鬼笔环肽免疫荧光染色发现细胞的丝状伪足增多,并且细胞内的应力纤维也变得粗壮而且排列有序;Transwell小室结果显示:跨膜细胞数随TGF-β_2浓度增加而逐渐增加,组间差异有统计学意义(F=37.45,P<0.05);E-cad mRNA和蛋白的表达随TGF-β_2的浓度增加逐渐下降,组间差异有统计学意义(P均<0.05);α-SMA、FN及Col-Ⅰ的mRNA和蛋白的表达随TGF-β_2浓度的增加而逐渐增高,组间差异均有统计学意义(P<0.05)。结论TGF-β_2可能通过促进RPE细胞发生EMT,导致Ⅰ型胶原蛋白和纤维连接蛋白等细胞外基质大量沉积,从而在增生性玻璃体视网膜病变(PVR)的发病过程中发挥重要作用。

Objective To study the effect of TGF-β_2on the epithelial mesenchymal transition of human retinal pigment epithelial cells.Methods Cultured ARPE-19 cells were treated with different concentrations of TGF-β_2（0,1,5,10ng/mL）for 24 h,the cell morphological changes were observed by immunofluorescence staining,the cell migration were observed by transwell chamber,the expressions of the epithelial marker of E-cad and the mesenchymal markers ofα-SMA,FN and Col-Ⅰ were detected by RT-PCR and Western blot method.ResultsWith the increase of the concentration of TGF-β_2,immunofluorescence staining of phalloidin showed that cell filopodia increased and stress fiber of cells became strong and orderly;The results of transwell chamber showed that with the increased concentration of TGF-β_2,the number of transmembrane cells increased significantly（F=37.45,P〈0.05）;The decreased expression of E-cad and the increased expressions ofα-SMA,FN and Col-Ⅰwere detected by RT-PCR and western blot gradually in both mRNA and protein levels significantly（P〈0.05）.Conclusion TGF-β_2 may play an important role in pathologic process of PVR by promoting the EMT of RPE cells and the accumulation of Fn and Col-Ⅰ.