Smad7对终末期糖基化产物介导大鼠肾小管上皮细胞转分化及Ⅰ型胶原合成的影响

Effects of Smad7 on transdifferentiation and collagen I synthesis in AGE-stimulated NRK52E cells

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 effects of smad7 on transdifferentiation and collagen Ⅰ synthesis in advanced glycosylation end-products （AGE）-stimulated NRK52E cells. Methods NRK52E cells were transferred by pTet-on plasmid system and the cell lines of doxycycline （Dox） -regulated Smad7 expression were selected for the study. Transnuclear location of p-Smad2/3 was examined with immunocytochemistry. The mRNA and protein expressions of Smad7, α-SMA, E-cadherin, collagen Ⅰ were detected with RT-PCR and Western blot. Results AGE-induced expressions of Smad7 mRNA and protein were further increased in NRK52E cells by the addition of Dox in a dose-dependent manner. Overexpression of Smad7 caused a marked inhibition of p-Smad2/3 transnuclear location at 30 rain （68.3% vs 31.2%, P 〈0.01 ） and 24 h （69.8% vs 28.7% , P 〈0.01 ）. Doxinduced overexpression of Smad7 inhibited AGE-induced α-SMA and collagen Ⅰ synthesis and up-regulated E- Cadherin mRNA and protein levels. Conclusion Overexpression of Smad7 can inhibit AGE-stimulated transdifferentiation and collagen Ⅰ production in NRK52E cells.