TGF-β1经Rho／ROCK信号途径诱导尿道瘢痕成纤维细胞表型转化及细胞外基质合成

TGF-β1 induces phenotypic differentiation and synthesis of extracellular matrix in fibroblasts derivedfrom urethral scar via Rho/ROCK signal pathway

目的探讨Rho／RCYCK信号通路是否参与了TGF-β1诱导的尿道瘢痕成纤维细胞表型转化及细胞外基质过度合成。方法进行原代培养人尿道瘢痕成纤维细胞，取第四代成纤维细胞用于实验。细胞达到80％融合时，培养液中加入TGF-β1（5、10ng／m1），培养48h后，用Western-blot检测各组a-SMA的变化；ELISA测定细胞Ⅲ型胶原及纤维结合素的表达；另用Rho／RoCK信号通路阻断剂Y-27632处理后，再用TGF-β1（10ng／m1）刺激，48h后，用Western-blot检测各组a-SMA的变化；ELISA测定细胞Ⅲ型胶原及纤维结合素的表达。结果TGF-β1能显著诱导α-SMA表达，且随TGF-β1浓度的升高其诱导作用呈逐渐增强趋势，TGF-β1同样能诱导Ⅲ型胶原及纤维结合素的表达（P〈0．01）。用Y27632（10“mol／L）处理后，再用TGF-β1刺激，α-SMA、Ⅲ型胶原及纤维结合素的表达显著受抑制（P〈0．01）。结论Rho／ROCK信号通路参与了TGF-β1诱导的尿道瘢痕成纤维细胞表型转化及细胞外基质过度合成，这为临床预防和治疗尿道瘢痕狭窄提供了理论依据。

Objective To examine the effect of Rho/ROCK signal pathway on TGF-β1- induced phenotypic differentiation and synthesis of extraeellular matrix in fibroblasts derived from urethral scar. Methods Fibroblasts derived from urethral scar were cultured. All experiments were performed using the cells at the fourth passage. At 80% confluence the medium was supplemented with TGF-β1（5,10 ng/ml）. After 48 hours incubation, the productions of α-SMA were assayed by Western -blot. The productions of collagen Ⅲ and fibronectin in supernatants culture were examined using ELISA. Then the fibrohlasts were stimulated with TGF-β1 （10 ng/ml） after treated with the Rho/ROCK signal pathway inhibitor Y-27632（10 μmol/L）. After 48 hours incubation, the productions of α-SMA, colla- gen Ⅲ, and fibronectin were assayed. Results TGF-β1 markedly induced α-SMA, collagen HI, and fi- bronectin expression in cultured fibroblasts in a dose-dependent manner （P〈0. 01）. However, simul- taneous incubation of Y-27632 significantly abrogated TGF-β1-induced α-SMA, collagen Ill, and fi- bronectin expression （P〈0. 01）. Conclusions Rho/ROCK signaling pathway should be involved in on TGF-β1-induced phenotypic differentiation and synthesis of extracellular matrix in fibroblasts derived from urethral scar. The findings provide a basis for prevention and treatment of urethral scar.