【摘要】 目的 探讨终末期糖基化终产物(AGEs)介导肾小管上皮细胞转分化和胶原 (Col)I合成的分子机制。方法 体外培养正常大鼠近端肾小管上皮细胞系(NRK52E),应用自 制的AGE-牛血清白蛋白(BSA)刺激NRK52E细胞。免疫细胞化学方法检测不同时间磷酸化(p) Smad2／3核转位情况。ELISA方法检测细胞培养上清TGF-β1的水平。RT-PCR方法检测α-平滑 肌肌动蛋白(SMA)、E-钙黏着糖蛋白(cadherin)和Col I mRNA表达。Western印迹检测α-SMA、 E-cadherin和Col I蛋白的表达。同时观察TGF-β1中和抗体对AGE-BSA上述效应的阻断作用。 结果 基础状态下,NRK52E细胞存在低水平p-Smad2／3核表达(16％)。与BSA对照组比较, AGE-BSA以时间依赖方式上调NRK52E细胞p-Smad2／3核转位,其高峰出现在30 min(68％比 30．5％,P<0．01)和24 h(76％比31．3％,P<0．01)。AGE-BSA显著上调α-SMA和Col I mRNA和 蛋白表达;下调E-cadherin mRNA和蛋白的表达;并能促进NRK52E细胞合成和分泌TGF-β1。 TGF-β1中和抗体能明显抑制AGE-BSA介导的24 h p-Smad2／3核转位(25．2％,P<0．01),但不 能阻抑30 min活化高峰;能明显抑制AGE-BSA介导的α-SMA和Col I mRNA和蛋白表达。以及 显著地上调E-cadherin mRNA和蛋白的表达。结论 AGEs通过TGF-β依赖和非依赖途径诱导 肾小管上皮细胞Smads信号通路活化,促进其向肌成纤维母细胞转分化和Col I的合成。更多还原

【Abstract】 Objective To investigate the molecular mechanisms that advanced glycation end products (AGE) stimulate epithelial-myofibroblast transdifferentiation and collagen I synthesis. Methods Advanced glycation end products was prepared by incubating bovine serum albumin (BSA) with D-glucose. Normal rat proximal tubular epithelial (NRK52E) cells were cultured in RPMI-1640 medium with AGE-BSA. Phosphorylation and nuclear translocation of Smad2/3 were examined by immunocytochemistry. Levels of TGF-B1 in supernatant of cell culture were measured by enzyme-linked immunosorbent assay (ELJSA). Expression of a-SMA, E-cadherin and collagen I mRNA was detected by RT-PCR. Expression of a-SMA, E-cadherin and collagen I proteins was detected by Western blot. AGE-BSA’s reaction to neutralizing TGF-B1 antibody was observed. Results AGE-BSA induced Smad2/3 phosphorylation and nuclear translocation, two peaks occured at 30 min (68% vs 30.5%,P < 0.01) and 24 h(76% vs 31.3%, P < 0.01), respectively, as compared to BSA group. The levels of TGF-B1 were markedly increased in supernatant of cell culture at 24 h and 48 h. AGE-BSA significantly up-regulated the expression of a-SMA, collagen I mRNA and proteins, whereas it down-regulated the expression of E-cadherin mRNA and protein. Neutralizing TGF-B1 antibody inhibited AGE-BSA-induced Smad2/3 peak reaction at 24 h (25.2% vs 76%, P < 0.01), and the expression of a-SMA, collagen I mRNA and proteins as well, whereas it up-regulated the expression of E-cadherin mRNA and protein. Conclusion AGEs induce activation of Smads signaling pathway, transdifferentiation and collagen I synthesis of proximal tubular epithelial cells via TGF-p-dependent and independent mechanisms.更多还原