Smad7对高糖介导大鼠肾小管上皮细胞转分化和胶原Ⅰ合成的影响

Effect of Smad7 on epithelial-myofibroblast transdifferentiation and collagen I synthesis in NRK52E cells stimulated by high glucose

目的探讨Smad7对高糖介导肾小管上皮细胞转分化和胶原(Col)Ⅰ合成的影响。方法体外培养转染Smad7的大鼠近端肾小管上皮细胞株(NRK52E细胞),分为强力霉素(Dox)诱导组和未加强力霉素的对照组。前者予Dox诱导24 h后,给予高糖刺激;后者不加Dox诱导。采用免疫细胞化学方法检测磷酸化(p)-Smad2/3核转位情况;RT-PCR检测Smad7的表达;Western印迹方法检测不同时间点Smad7、α-SMA、E-钙黏蛋白(cadherin)和ColⅠ蛋白的表达水平。结果成功构建了Dox调控的可上调Smad7表达的NRK52E细胞。NRK52E细胞在基础状态下可表达低水平p-Smad2/3(16．1％),与未加Dox的对照组比较,Dox诱导组可显著抑制高糖刺激的NRK52E细胞TGF-β受体调控信号蛋白Smad2/3的活化和核转位(30．3％比58．5％, P<0．01)。上调表达Smad7可显著抑制高糖介导的NRK52E细胞α-SMA和ColⅠ蛋白的表达;逆转高糖介导的NRK52E细胞E-cadherin蛋白的下调表达。结论基因转染上调表达Smad7可通过TGF-β受体调控信号蛋白Smad2/3的活化和核转位而阻抑高糖介导的肾小管上皮细胞转分化及细胞外基质的合成。

Objective To investigate the effect of Smad7 on epithelial-myofibroblast transdifferentiation and collagen I synthesis in NRK52E cells stimulated by high glucose. Methods Smad7-expression NRK52E cells were grown in RPMI 1640 medium containing 10% fetal bovine serum. They were cultured for 24 h in free serum medium as well as 60%-80% cells were adhered onto the surface of the flask. Afterwards, Smad7-expression NRK52E cells were incubated in the presence or absence of doxycycline （Dox, 2 μg/ml） for 24 h, then stimulated by high glucose, p-Smad2/3 nuclear translecation induced by high glucose was examined by immunocytochemistry. The expression of Smad7 mRNA was detected by RT-PCR. The expression of Smad7, α-SMA, E-cadherin and Col I protein was detected by western blot. Results Smad7-expression NRK52E cells were regulated by Dox compared with the smad7 expression level in no Smad7-transfected NRK52E cells. Smad7 mRNA and protein were highly expressed in smad7 gene-transfected NRK52E cells induced by high glucose. The expression level of p-Smad2/3 was low in normal NRK52E cells （16.1%）. Overexpression of Smad7 markedly suppressed the p-Smad2/3 expression and nuclear tranalecation （30.3% vs 58.5%, P 〈 0.01 ）induced by high glucose. Up-regulated expression of Smad7 inhihited the high expression of R-SMA and Col I protein and reversed E-cadherin protein expression level induced by high glucose. Conclusion Overexpression of Smad7 can suppress epithelial-myofibroblast transdifferentiation and collagen I synthesis induced by high glucose through blocking Smad2/3 activation.