论文部分内容阅读
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 investigate whether Smad signal transduction pathway in human peritoneal mesothelial cells plays a role in peritoneal fibrosis induced by TGF β1. Methods: Primary human peritoneal mesothelial cells were obtained from healthy adult omentum. More than 95% human peritoneal mesothelial cells were identified. The third generation cultured cells were stimulated with 5ng / ml TGFβ1, and the protein expression of phosphorylated Smad2 / 3 (p Smad2 / 3) in human peritoneal mesothelial cells was observed by immunohistochemical staining, Westernblotting, ELISA and RT PCR, respectively Intracellular migration; intracellular Smad7 protein and mRNA expression; extracellular fibronectin (FN) protein, intracellular FN mRNA, and intracellular type I collagen (COL1) protein and mRNA expression. Results: The expression of p Smad2 / 3 protein in human peritoneal mesothelial cells began to increase at 15 min after TGF β1 stimulation. At this time, the positive rate of p Smad2 / 3 was 29%. The staining of cells was mainly in the cytoplasm, 30min positive rate of 81% to 1h 84% of the positive cells increased most significantly, the cells deepened and concentrated in the nucleus and surrounding nuclei, 2h protein expression decreased significantly, then the positive cell rate was 37%, the cells stained lighter and dispersed to The expression of Smad7 in the cytoplasm increased significantly at 24 hrs after TGFβ1 stimulation and peaked at 48 hrs, and the mRNA expression was enhanced in time-dependent manner. The expression of extracellular FN, intracellular FN mRNA and intracellular COL1 And mRNA expression significantly increased from 24 h after TGF β1 stimulation, and all showed a certain degree of time compliance. Conclusion: Smad signal transduction pathway in human peritoneal mesothelial cells can?