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目的探讨Smad7对终末期糖基化产物(AGE)介导肾小管上皮细胞转分化及Ⅰ型胶原合成的影响。方法Tet-on质粒系统构建表达Smad7的正常大鼠近端肾小管上皮细胞(NRK52E)株,通过强力霉素(Dox)上调Smad7的表达,以细胞免疫化学、RT-PCR或Western印迹技术观察Smad7对AGE介导NRK52E细胞磷酸化(p)Smad2/3核转位、及α-平滑肌肌动蛋白(SMA)、E-钙黏着糖蛋白(cadherin)和Ⅰ型胶原mRNA和蛋白表达的影响。结果Smad7转染的NRK52E细胞以剂量依赖方式受Dox调控。Smad7的高表达可抑制AGE介导的30 min(68.3% vs 31.2%,P<0.01)和24h(69.8% vs 28.7%,P<0.01)p- Smad2/3的核转位水平;明显下调α-SMA、Ⅰ型胶原mRNA和蛋白的表达,上调E-钙黏着糖蛋白mRNA和蛋白的表达。结论Smad7通过抑制Smad信号通路活化,阻抑AGE介导的肾小管上皮细胞向肌成纤维母细胞转分化和Ⅰ型胶原的合成。
Objective To investigate the effect of Smad7 on the transdifferentiation of renal tubular epithelial cells and the synthesis of type Ⅰ collagen induced by terminal glycation end products (AGE). Methods The normal rat proximal tubular epithelial cells (NRK52E) expressing Smad7 were constructed by Tet-on plasmid system. The expression of Smad7 was up-regulated by doxorubicin (Dox), and Smad7 was detected by immunocytochemistry, RT-PCR or Western blotting On AGE-mediated phosphorylation of p-Smad2 / 3 nuclear translocation and the expression of α-smooth muscle actin (SMA), E-cadherin and collagen type Ⅰ mRNA and protein in NRK52E cells. Results Smad7-transfected NRK52E cells were Dox regulated in a dose-dependent manner. High expression of Smad7 could inhibit AGE-mediated nuclear translocation of p-Smad2 / 3 for 30 min (68.3% vs 31.2%, P <0.01) and 24h (69.8% vs 28.7%, P <0.01) -SMA, type Ⅰ collagen mRNA and protein expression, increased E-cadherin protein mRNA and protein expression. Conclusion Smad7 inhibits the AGE-mediated transdifferentiation of renal tubular epithelial cells into myofibroblasts and the synthesis of type I collagen by inhibiting the Smad signaling pathway.