TGF-β1对大鼠腹膜间皮细胞转分化的影响及其机制

Effect of TGF-β1 on epithelial-mesenchymal transition of rat peritoneal mesothelial cells and its mechanism

目的:研究转化生长因子(transforming growth factorβ1,TGF-β1)对大鼠腹膜间皮细胞(rat peritoneal mesothelial cells,RPMCs)转分化的影响,并探讨其可能的机制。方法:体外培养SD大鼠原代腹膜间皮细胞,静止24h后,随机分为正常对照组和TGF-β1(10μg/L)刺激组。用TGF-β1(10μg/L)刺激RPMCs不同时间,分别用RT-PCR方法和Western免疫印迹法检测α-平滑肌肌动蛋白(α-smooth muscleactin,α-SMA)、E-钙黏素、Ⅰ型胶原的mRNA和蛋白的表达。用Western免疫印迹法检测总RhoA的蛋白表达水平,活化的RhoA由膜蛋白提取试剂盒提取,并用Western免疫印迹法评估。结果:TGF-β1刺激RPMCs能导致E-钙黏素mRNA和蛋白表达下调;α-SMA,Ⅰ型胶原mRNA和蛋白表达上调,同时RhoA蛋白表达上调,呈时间依赖性。结论:TGF-β1诱导了大鼠腹膜间皮细胞转分化,其机制可能通过RhoA介导的信号通路。

Objective To explore the effect of transforming growth factor β1 （TGF-β1） on epithelial-mesenchymal transition in rat peritoneal mesothelial cells （RPMCs） and its mechanism. Methods Primary peritoneal mesothelial cells of SP rats were cultured in vitro. After synchronization for 24 h, RPMCs were randomly divided into 2 groups ： Group A （control） , Group B （ TGF-β1 , 10 μg/L）. RPMCs were stimulated by 10 μg/L TGF-β1 for different time. The mRNA and protein expression levels of E-cadherin, u-smooth muscle actin （α-SMA） and collagen 1 were measured by RT-PCR and Western blot, respectively. The protein expression level of total RhoA was measured by Western blot. Active RhoA was extracted by Plasma Membrane Protein Extraction Kit, and assessed by Western blot. Results TGF-β1 down-regulated mRNA and protein expression of E-cadherin in RPMCs, and upregulated mRNA and protein expression of α-SMA and Collagen Ⅰ . TGF-β1 stimulation elicited a robust increase in RhoA activity in a time-dependent manner. RhoA activity peaked at 1 h. Conclusion RPMCs can be transdifferentiated into myofibroblast under the effect of TGF-β1, and the mechanism may be related to the activation of RhoA associated signal pathway.