肝细胞生长因子下调Smurf2拮抗肾小管上皮细胞-间充质细胞转分化  

Hepatocyte growth factor surpresses epithelial-mesenchymal transition through down- regulating Smurf2 expression in rat NRK-52E cells

作　　者：谭若芸[1] 方奕[1] 苏卫芳[1] 杨俊伟[1] 张炜[2] 顾民[2] 

机构地区：[1]南京医科大学第二附属医院肾脏病中心,210003 [2]江苏省人民医院泌尿外科

出　　处：《中华肾脏病杂志》2012年第8期616-621,共6页Chinese Journal of Nephrology

基　　金：江苏省自然科学基金项目（BK2008482）;中华医学会临床医学科研专项基金（09010310186）

摘　　要：目的通过观察肝细胞生长因子（HGF）对正常大鼠肾上皮细胞（NRK-52E）转化生长因子p1（TGF—B1）诱导的Smad泛素化调节因子2（Smurf2）表达的影响，探讨HGF拮抗肾小管上皮细胞-间充质细胞转分化（EMT）的分子机制。方法以NRK。52E细胞为研究对象，给予TGF—B1（5μg/L）处理0～24h；或部分细胞经HGF（20μg／L）预处理30min后，再予TGF-β1（5μg，L）处理1h或48h；另外部分细胞予以Smurf2质粒表达载体或Smurf2siRNA转染24h后，再予HGF处理24h。应用Western印迹及间接免疫荧光染色方法检测Smurf2、SnoN、E钙黏蛋白（E—cadherin）、α平滑肌肌动蛋白（d—SMA）和纤连蛋白（FN）的表达。结果与对照组相比，TGF—β1处理后迅速上调NRK-52E细胞中Smurf2蛋白表达（P〈0．01）；同时显著诱导FN和α—SMA蛋白表达，并下调E-cadherin表达。而予HGF预处理细胞后，可快速抑制TGF—β1诱导的Smurf2表达上调（P〈0．01）；并逆转TGF-β1介导的SnoN（P〈0．01）、E-cadherin（P〈0．05）、α-SMA（P〈0．01）和FN（P〈0．01）表达变化。此外，在NRK-52E细胞中过表达Smurf2蛋白可部分抑制HGF诱导的SnoN蛋白上调；而抑制Smurf2表达则可进一步促进HGF诱导的SnoN蛋白表达。结论在肾小管上皮细胞中，HGF可能通过下调Smurf2表达抑制SnoN蛋白发生泛素-蛋白酶体依赖性降解，进而拮抗TGF-β1介导EMT形成。Objective To investigate the possible mechanism that hepatocyte growth factor （HGF） inhibits renal tubular epithelial-mesenchymal transition （EMT）, and to determine whether Smurf2 expression induced by TGF-β1 can be reversed by HGF in normal rat kidney epithelial cells （NRK-52E）. Methods Using rat NRK-52E cell line as an in vitro system, NRK-52E cells were incubated with 5 μg/L TGF-β1 for 0-24 h. Part of cells were pretreated with 20 μg/L HGF for 30 min or not, then incubated with or without 5μg/L TGF-β1 for 1 h or 48 h. The other cells were transfected with pFlag-Smurf2 or Smurf2 siRNA for 24 h, then treated with or without 20 μg/L HGF for 24 h. The expressions of Smurf2, SnoN, E-cadherin, alpha-smooth muscle actin （α-SMA） and fibronectin （FN） were detected by Western blotting and indirect immunofluorescence staining assays. Results Compared to normal control, TGF-β1 could rapidly induce Smurf2 protein expression in a short time （P〈0.01）. Meanwhile, the expressions of FN and α-SMA were significantly induced, and the expression of E-cadherin was reduced in NRK-52E cells by TGF-β1. In contrast, in the NRK-52E cells pretreated with HGF, HGF could obviously inhibit Smurf2 expression induced by TGF-β1, and reversed the down-regulation of SnoN （P〈0.01） and E-cadherin （P〈0.05）, the up-regulation of oL-SMA （P〈0.01） and FN （P〈0.01） induced by TGF-β1. Moreover, overexpression of Smurf2 in NRK-52E cells could partly inhibit the up-regulation of SnoN protein by HGF, while down-regulation of Smurf2 could up-regulate the expression of SnoN induced by HGF. Conclusions HGF can abolish EMT induced by TGF-β1 in renal tubular epithelial cells through down-regulating Smurf2 expression and suppressing ubiquitin-proteasome dependent degradation of SnoN.

关 键 词：肝细胞生长因子 纤维化 转化生长因子Β1 Smad泛素化调节因子2 上皮细胞.间充质细胞转分化 

分 类 号：R363[医药卫生—病理学]