摘要
【目的】观察TGF β1对人腹膜间皮细胞 (HPMCs)纤溶酶原激活物抑制剂 1(PAI 1)的mRNA和蛋白表达影响并就可能的机制进行探讨。【方法】取健康成年人大网膜进行原代培养 ,用 5ng/mlTGF β1刺激第三代培养细胞 ,采用免疫组织化学染色、Westernblot、ELISA以及RT PCR等方法 ,分别观察PAI 1的mRNA和蛋白表达 ,纤维连接蛋白 (FN)和Ⅰ型胶原 (COLⅠ )的mRNA和蛋白表达 ,细胞内磷酸化Smad2 / 3(p Smad2 / 3)的蛋白表达。【结果】刺激组PAI 1的mRNA和蛋白表达与对照组相比均显著增加 ,其中 2 4h为高峰 ,4 8h、72h依次递减 ;刺激组FN和COLⅠ的mRNA和蛋白表达与对照组比均呈时间依从性显著增加 ;对照组细胞内几乎不表达p Smad2 / 3,阳性细胞率 3% ,在刺激后 15min蛋白表达即开始增加 ,阳性细胞率2 9 % ,1h增加最明显 ,阳性细胞率达 84 % ,2h明显回落 ,此时阳性细胞率为 37%。【结论】TGF β1在致腹膜纤维化过程中诱导了HPMCs内PAI 1的转录与蛋白表达 ,这可能与TGF β1激活了HPMCs内Smad信号通路有关。
Objectives]To observe the effects of transforming growth factor-β 1(TGF-β 1) stimulation on the mRNA and protein expressions of plasminogen activator inhibitor-1 (PAI-1) in human peritoneal mesothelial cells (HPMCs) and explore its possible mechanism of action.Omentum majus of healthy adult was used to culture HPMCs in primary generation, the third generation of cultured cells was stimulated by 5 ng/ml TGF-β 1. Immunohistochemical staining, Western blotting, ELISA and RT-PCR were employed to observe the mRNA and protein expressions of PAI-1, mRNA and protein expressions of fibronectin (FN) and type I collagen (COLⅠ) as well as the protein expression of p-Smad 2/3 and its migration in HPMCs.The mRNA and protein expressions of PAI-1 in TGF-β 1 group significantly increased as compared to those in control group, reaching to their peaks at 24h then declining gradually from 48 h to 72 h. The mRNA and protein expressions of FN and COLⅠ in TGF-β 1 group were significantly higher than those in the control group, and this increase displayed time dependency. p-Smad2/3 almost not expressed in HPMCs of the control group, with only a positive cell rate of 3%. Its expression began to raise 15 min after TGF-β 1 stimulation (positive cell rate 29%), became most prominent at 1 h (84%) and dropped markedly 2 h later (37%).[Conclusion]TGF-β 1 can induce the mRNA and protein expression of PAI-1 within HPMCs in the process of human peritoneal fibrosis. Its possible mechanism is related with the activation of Smad signal pathway in HPMCs through TGF-β 1 stimulation.
出处
《医学临床研究》
CAS
2004年第8期864-867,873,共5页
Journal of Clinical Research
