【摘要】 目的　探讨TGF - β1介导的Smad信号通路对人腹膜间皮细胞 (HPMCs)细胞外基质 (ECM)的调控机制。方法　原代培养的第三代HPMCs分成对照组与 5ng/mlTGF - β1刺激组 ,采用免疫组织化学染色和Western印迹法观察细胞内磷酸化Smad2 / 3(p -Smad2 / 3)的表达以及在细胞内的迁移 ,Western印迹、ELISA和RT -PCR法观察Smad7、结缔组织生长因子 (CTGF)、α-平滑肌肌动蛋白 (α-SMA)、纤溶酶原激活物抑制剂 -1(PAI- 1)、纤维连接蛋白 (FN)和I型胶原 (COL1)的mRNA及蛋白表达。结果　①对照组细胞内几乎不表达p -Smad2 / 3,在刺激后 15min蛋白表达增加 ,主要分散在细胞质中 ,1h达高峰 ,主要集中在细胞核及周边 ,2h明显回落 ,又分散至细胞质中 ;②刺激组细胞内Smad7、CTGF、α -SMA和COL1的蛋白表达与对照组比均增加 ,其中Smad7、CTGF和α -SMA在 4 8h最明显 ,COL1呈时间依从性 ;刺激组上清液PAI - 1的蛋白含量与对照组比均增加 ,其中 2 4h最高 ,FN呈时间依从性 ;刺激组Smad7的mRNA表达与对照组比呈时间依从性增加 ,CTGF、α -SMA、PAI- 1、FN和COL1均增加 ,其中CTGF和α-SMA在 4 8h最高 ,PAI- 1在 2 4h最高 ,FN和COL1呈时间依从性。结论　TGF - β1能特异性激活HPMCs内Smad信号通路 ,并可通过诱导该通路下游靶基因Sm更多还原

【Abstract】 Objective To investigate the regulatory mechanism of Smad signaling pathway mediated by TGF-β 1 on extracellular matrix (ECM) of human peritoneal mesothelial cells (HPMCs). Methods The third generation HPMCs from primary culture were divided into the control group and the treatment groups with 5 ng/ml TGF-β 1 stimulation. Immunohistochemistry and Western blot assay were used to detect the expression of p-Smad2/3 and its migration in cells. Western blot assay, ELISA and semi-quantification RT-PCR were used to detect the mRNA and protein expressions of Smad7, connective tissue growth factor (CTGF), α- smooth muscle actin (α-SMA), plasminogen activator inhibitor-1(PAI-1), fibronectin (FN) and collagen, type I (COL1). Results ①p-Smad2/3 in HPMCs was also almost not expressed in the control group, and remarkably increased 15 min after TGF-β 1 stimulation, peaking at 1 h and dropping after 2h; Meanwhile, p-Smad2/3 mainly distributed in cytoplasm at 15 min, concentrated in cell nucleus and peri-nucleus at 1 h, and distributed in cytoplasm again at 2h; ② The protein expressions of intracellular Smad7, CTGF, α-SMA and COL1 in the treatment groups were more obviously increased than those in the control group, peaking at 48 h such as Smad7, CTGF and α-SMA, and increased in time-dependent manner such as COL1; The protein contents of supernatant PAI-1 in the treatment groups were more significantly increased than that in the control group(24h、48h P<0.01,72 h P<0.05), peaking at 24 h, and the protein contents of supernatant FN in the treatment groups were more remarkably increased than that in the control group in a time-dependent manner (P<0.01);The mRNA levels of Smad7 in the treatment groups were more significantly increased than that in the control group in a time-dependent manner (24h P<0.05,48 h、72 h P<0.01), and the mRNA levels of CTGF、α-SMA、PAI-1、FN and COL1 in the treatment groups were more remarkably increased than those in the control group (P<0.01), peaking at 48 h such as CTGF and α-SMA, peaking at 24 h such as PAI-1, and increased in a time-dependent manner such as FN and COL1. Conclusion TGF-β 1 can specifically activate Smad signaling pathway in HPMCs, and participates in the regulatory effects of ECM by inducing the transcription and expression of some downstream target genes such as Smad7、CTGF、α-SMA、PAI-1、FN and COL1 of Smad signaling pathway.更多还原