TGF-β1刺激对人腹膜间皮细胞内Smad信号转导通路的影响

目的：探讨人腹膜间皮细胞内Smad信号转导通路在TGF-β1致腹膜纤维化过程中是否发挥作用。方法：取健康成年人大网膜进行人腹膜间皮细胞原代培养，经鉴定95%以上为人腹膜间皮细胞。用5 ng/ml TGF-β1刺激第三代培养细胞，采用免疫组织化学染色、Western blotting，ELISA以及RT-PCR等方法，分别观察人腹膜间皮细胞内磷酸化Smad2/3（p-Smad2/3）的蛋白表达以及在细胞内的迁移；细胞内Smad7的蛋白和mRNA表达；细胞外纤维连接蛋白（FN）的蛋白、细胞内FN的mRNA以及细胞内I型胶原（COL1）的蛋白和mRNA表达。结果：人腹膜间皮细胞内p-Smad2/3的蛋白表达在TGF-β1刺激后15 min即开始显著增加，此时p-Smad2/3的阳性细胞率为29%，细胞着色主要分散在细胞质中，30 min阳性细胞率81%至1 h阳性细胞率84%增加最明显，细胞着色加深且集中在细胞核及周边，2 h蛋白表达明显回落，此时阳性细胞率为37%，细胞着色转淡并又分散至细胞质中；细胞内Smad7的蛋白表达在TGF-β1刺激后24 h明显增加，48 h时达高峰，mRNA表达呈时间依从性增强；细胞外FN的蛋白、细胞内FN的mRNA以及细胞内COL1的蛋白和mRNA表达从TGF-β1刺激后24 h起均明显增加，且均显示一定的时间依从性。结论：人腹膜间皮细胞内Smad信号转导通路能被TGF-β1特异性激活，并在TGF-β1致腹膜纤维化过程中发挥效应；经TGF-β1刺激后，在该通路中主要起抑制作用的Smad7蛋白和mRNA表达均反馈性增加。

Effect of TGF-β1 stimulation on the Smad signal transduction pathway of human peritoneal mesothelial cells

OBJECTIVE: To explore whether the Smad signal transduction pathway in human peritoneal mesothelial cells (HPMCs) influences the process of human peritoneal fibrosis stimulated by TGF-beta1. METHODS: HPMCs were isolated from normal human omentum and 95% of the primary cultured cells was confirmed to be HPMCs. The third generation of cultured cells was stimulated by 5 ng/ml TGF-beta1. Immunohistochemistry, Western blotting, ELISA, and RT-PCR were employed to investigate the follows: the protein expression of p-Smad2/3 and its migration in HPMCs; the protein and mRNA expressions of SMAD 7 in cells; and the expressions of extracellular fibronectin (FN) protein, intracellular FN mRNA, as well as intracellular collenge-I (COL1) protein and mRNA. RESULTS: The protein expression of p-Smad2/3 in HPMCs obviously increased 15 min (29% p-Smad2/3-positive cells) after TGF-beta1 stimulation, peaking from 30 min (81% ) to 1 h (84%) and dropping after 2 h (37%); Meanwhile, p-Smad2/3 mainly distributed in cytoplasm at 15 min, concentrated in cell nucleus and peri-nucleus from 30 min to 1 h, and distributed in cytoplasm again at 2 h. The protein expression of SMAD7 in cells obviously increased 24 h after TGF-beta1 stimulation, peaking at 48 h. The mRNA expression of SMAD7 time-dependently increased. The expressions of extracellular FN protein, intracellular FN mRNA, as well as intracellular COL1 protein and mRNA significantly increased and all of them displayed time dependency. CONCLUSION: The SMAD signal transduction pathway of HPMCs can be specifically activated by TGF-beta1 and influence the process of human peritoneal fibrosis. With the stimulation of TGF-beta1, the protein and mRNA expressions of SMAD 7 (an inhibitor of SMAD pathway) significantly increase as a result of feedback.