高糖通过转化生长因子β1-Smad信号传递途径诱导肾小管上皮细胞转分化  

High glucose induces renal epithelial-mesenchymal transition through transforming growth factor βl-Smad signaling pathway

作　　者：张飞飞 潭若云 熊明霞 王晓华 方丽 樊伟 王笑云 杨俊伟 

机构地区：[1]南京医科大学第一附属医院肾脏病科、江苏省糖尿病临床医学中心,210029

出　　处：《中华肾脏病杂志》2008年第3期174-178,共5页Chinese Journal of Nephrology

基　　金：国家自然科学基金（30470800、30771010）;科技部“973”重点项目（2006CB503909）;江苏省“科教兴卫”工程医学领军人才项目

摘　　要：目的通过观察高糖对肾小管上皮细胞转分化（EMT）的作用，探讨其与转化生长因子131（TGF-131）的关系及糖尿病肾病肾小管间质纤维化的发病机制。方法以人肾小管上皮细胞株HKC细胞和高表达Smad7蛋白的HKC转染细胞株为研究对象。蛋白印迹法检测高糖（葡萄糖浓度分别为25和50mmol／L）对α平滑肌肌动蛋白（α-SMA）、E钙黏蛋白（E-cadherin）和纤连蛋白（FN）表达的影响。酶联免疫吸附（ELISA）法检测TGF-1l的水平。Boyden小室检测HKC细胞的迁移能力。抗TGF-β1抗体中和实验分析高糖对肾小管EMT作用及与TGF-β1的关系。结果持续的高糖作用（96h）能够导致HKC表达α-SMA蛋白，其中25和50 mmol/L的葡萄糖分别增加表达α-SMA 2．8倍和8．2倍；降低E-cadherin的表达；刺激合成FN。25和50mmol／L的葡萄糖刺激HKC 12h后，细胞培养上清液中TGF-β1浓度分别为（408．5±198．6）和（939．3±311．8）ng／L，呈剂量依赖性。抗TGF-βl抗体能够显著抑制高糖导致的HKC高表达α-SMA蛋白和FN及降低E-cadherin表达的作用。高表达Smad7蛋白的HKC转染细胞株在高糖的持续作用下，不能表达α-SMA和FN蛋白，E-cadherin也未见降低。细胞迁移实验表明，25和50mmol／L高糖能够增加HKC迁移至Boyden小室膜下侧面的细胞数[（12．4±3．7）和（18．6±4．4）细胞／HP）]，与正常对照组[（3．0±0．8）细胞／HP]差异有统计学意义（P〈0．01）。抗TGF-β1多克隆抗体能够部分抑制高糖（50mmol／L）造成的HKC细胞向Boyden小室膜下侧面的迁移[（11．9±5．2）细胞／HP]。高表达Smad7蛋白的HKC转染细胞株在高糖培养条件下迁移至Boyden小室膜下侧面的细胞数[（4．3＋1．2）细胞／HP]与正常对照组差异无统计学意义。结论高糖能够诱导肾小管EMT，此作用与高糖刺激该细胞合成TGF-β1有关，阻止Smad信号途径能够拮抗TGF-β1介导的肾小管EMT的作用�Objective To investigate the effect of high glucose on renal tubular epithelial-mesenchymal transition, and to analyze the relationship between high glucose and transforming growth factor [β（TGF-β1） and the mechanism of renal interstitial fibrosis. Methods HKC and Smad7-overexpression HKC cells were grown in DMEM/F12 medium containing 5%-10% newborn calf serum. They were cultured for 16 h in free serum medium after 80% cells were adhered onto the surface of the flask.Afterwards,they were stimulated by high glucose （glucose concentration：25 mmol/L and 50 mmol/L）.The expression of α-SMA,E-cadherin and fibronectin was detected by Westem blot while the supematant level of TGF-β1 was detected by ELISA.Cell motility and migration was evaluated using Boyden chamber motogenicity assay. Results In HKC induced by high glucose,the expression of α-SMA and fibronectin protein was highly up-regulated while the expression of E-cadherin protein was down-regulated.The expression of TGF-β1 was up-regulated in a dose-dependent manner.These above-mentioned effects could be obviously inhibited by anti-TGF-β1 antibody.The protein HKC induced by high glucose.HKC exhibited enhanced motility and invasive capacity in high glucose groups, compared to that in control group.Migrated cell counting was （12.4±3.7） and （18.6±4.4） cell/HP in 25 and 50 mmol/L glucose groups respectively. Conclusion High glucose may induce renal tubular epithelial- mesenchymal transition through TGF--β1 pathway, which can be inhibited by blocking the Smad signal pathway.

关 键 词：转化生长因子Β 糖尿病肾病 肾小管 SMAD 上皮细胞转分化 

分 类 号：R692[医药卫生—泌尿科学]