中国血液净化

›› 2004, Vol. 3 ›› Issue (11): 609-614.

• 基础研究 • 上一篇    下一篇

转化生长因子β1介导的Smad信号通路对人腹膜间皮细胞外基质的调控

刘伏友 张 浩 彭佑铭 刘映红 龙志高 吴鼎文 敖 翔 陈仙花   

  1. 410011 长沙，中南大学湘雅二医院肾内科（刘伏友，彭佑铭，刘映红）410013 长沙，中南大学湘雅三医院肾内科（张浩，博士研究生）410078 长沙，中南大学遗传学国家重点实验室（龙志高，吴鼎文，敖翔，陈仙花）
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2004-11-12 发布日期:2004-11-12

  • Received:1900-01-01 Revised:1900-01-01 Online:2004-11-12 Published:2004-11-12

RichHTML

0

PDF

180

摘要：

目的 探讨TGF-β1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/ml TGF-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15 min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48 h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24 h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48 h最高，PAI-1在24 h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Smad7 、CTGF、α-SMA、PAI-1、FN和COL1的转录与表达参与对ECM的调控。

关键词: 转化生长因子β1, 间皮细胞, Smad, 细胞外基质, 纤维化

Abstract:

Objective To investigate the regulatory mechanism of Smad signaling pathway mediated by TGF-β1 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-β1 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-β1 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<0.01，72h 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<0.05，48h、72h 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-β1 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. 

Key words: Mesothelial cells, Smad, Extracellular matrix, Fibrosis 

中图分类号: