TGF-β1刺激对人腹膜间皮细胞内Smad信号转导通路的影响

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

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 5ng/mlTGF β1刺激第三代培养细胞 ,采用免疫组织化学染色、Westernblotting ,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 1 5min即开始显著增加 ,此时p Smad2 / 3的阳性细胞率为 2 9% ,细胞着色主要分散在细胞质中 ,30min阳性细胞率 81 %至 1h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 ,2h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 2 4h明显增加 ,4 8h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 2 4h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?

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 β1.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 β1.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 β1 stimulation,peaking from 30 min (81%) to 1h (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 β1 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 β1 and influence the process of human peritoneal fibrosis.With the stimulation of TGF β1,the protein and mRNA expressions of SMAD 7 (an inhibitor of SMAD pathway) significantly increase as a result of feedback.