摘要目的 探讨RhoA-Rock信号通路在转化生长因子β1(TGF-β1)诱导大鼠腹膜间皮细胞(RPMC)转分化中的作用.方法 体外培养SD大鼠原代腹膜间皮细胞,静止24 h后,采用随机数字表法随机分为以下4组:正常对照组、TGF-β1(10μg/L)刺激组、TGF-β(10μg/L)+Y-27632(Rock特异性抑制剂,10 μmol/L)组(Y-27632预处理2 h)、Y-27632(10 μmol/L)组.用TGF-β1(10 μg/L)刺激RPMC不同时间,观察α平滑肌肌动蛋白(α-SMA),E钙黏素(E-cadhetin)、Ⅰ型胶原(Col Ⅰ)的表达.RT-PCR法检测E-cadherin、α-SMA和Col Ⅰ mRNA表达.Western印迹法检测RhoA(包括总RhoA及活化的RhoA)、E-cadhefin、α-SMA、Col Ⅰ和波形蛋白(vimentin)表达.活化的RhoA由膜蛋白提取试剂盒提取.结果 (1)TGF-β1(10 μg/L)刺激RPMC能诱导RhoA活化,于10 min开始出现活性升高,为对照组的(2.57±0.52)倍(P<0.05);1 h达高峰,为对照组的(4.35±0.41)倍(P<0.05).(2)TGF-β1(10 μg/L)刺激RPMC能导致E-cadherin mRNA和蛋白表达下调,α-SMA、Col Ⅰ mRNA和蛋白表达上调,呈时间依赖性.(3)Rock特异性抑制剂Y-27632能显著下调α-SMA、Col Ⅰ mRNA的表达,较TGF-β1刺激组各降低了53.8%和55.7%(均P<0.05),并且能下调α-SMA、Col Ⅰ和vimentin蛋白的表达,较TGF-β1刺激组分别降低了42.6%、60.1%和58.1%(均P<0.05),但不能上调E-cadherinmRNA和蛋白的表达.结论 TGF-β1可通过RhoA-Rock信号通路介导大鼠腹膜间皮细胞转分化,抑制该通路可作为防治腹膜纤维化的潜在靶点.

abstractsObjective To investigate the role of RhoA-Rock signaling pathway in the process of rat peritoneal mesothelial cells (RPMCs) epithelial-mesenchymal transition (EMT) induced by transforming growth factor β1 (TGF-β1). Methods Primary RPMCs were cultured in vitro. After synchronization for 24 hours, RPMCs were randomly assigned to 4 groups: group A (control), group B (TGF-β1, 10 μg/L), group C (10 μg/L TGF-β1+10 μmol/L Y-27632, an inhibitor of Rock, pretreated for 2 hours with Y-27632 before TGF-β1 stimulation), group D (Y-27632 alone, 10 μmol/L). Growth arrested and synchronized RPMCs were stimulated by 10 μg/L TGF-β1 for different time. The mRNA and protein expression levels of E-eadherin, α-SMA and collagen Ⅰ were measured by RT-PCR and Western blotting respectively. The protein expression level of vimentin was measured by Western blotting. Active RhoA was extracted by Plasma Membrane Protein Extraction Kit, then it was assessed by Western blotting. Results (1) TGF-β1 stimulation elicited a robust increase in RhoA activity in time-dependent manner, which was (2.57±0.52) folds compared with control group (P<0.05) after 10 min stimulation. RhoA activity peaked at 1 hour, which was (4.35±0.41) folds compared with control group (P<0.05). (2) TGF-β1 up-regulated mRNA and/or protein expression of α-SMA, vimentin and collagen Ⅰ , and down-regnlated mRNA and protein expression of E-cadherin in RPMCs. (3) The Rock inhibitor Y-27632 effectively revered TGF-β1-induced expression of α-SMA, collagen Ⅰ and vimentin. The mRNA levels of α-SMA and collagen Ⅰ decreased by 53.8% and 55.7%, and the protein levels of α-SMA, vimentin and collagen Ⅰ decreased by 42.6%, 60.1% and 58.1% compared with TGF-β1-stimulated groups (P< 0.05). But Y-27632 had no effect on the level of E-cadherin. Conclusions RhoA-Bock signaling pathway may mediate EMT induced by TGF-β1 in rat peritoneal mesothelial cells. RhoA-Rock pathway may be the potential therapeutic target in the progress of peritoneal fibrosis.