Smad4慢病毒转染对万古霉素诱导的肾小管上皮细胞EMT的影响

Effect of Smad4 Lentivirus Transfection on Vancomycin-induced EMT in Renal Tubular Epithelial Cells

目的:探究慢病毒介导过表达Smad4或沉默Smad4对万古霉素诱导的肾小管上皮细胞HK-2上皮间质转化(EMT)的影响。方法:设计并包装Smad4沉默或过表达的慢病毒,之后利用所获得的慢病毒瞬时转染HK-2,将HK-2细胞分为万古霉素组、空载体组(转染HBLV-GFP-PURO)、过表达Smad4组(转染HBLV-h-Smad4-GFP-PURO)、沉默Smad4组(转染HBLV-h-Smad4-shRNA-GFP-PURO),另取正常HK-2细胞作为对照组。比较五组细胞迁移能力、上清液中氧化应激指标(TGF-β_(1)、SOD、GSH、MDA)及Smad4、E-cadherin、FN、Vimentin蛋白表达水平。结果:与对照组相比,万古霉素组和空载体组HK-2细胞迁移能力、上清液TGF-β_(1)和MDA含量、Smad4、FN、Vimentin蛋白表达水平升高(P<0.05),SOD、GSH含量、E-cadherin蛋白表达水平降低(P<0.05)。过表达Smad4可提高HK-2细胞迁移能力和氧化应激反应,促进EMT(P<0.05);沉默Smad4可降低HK-2细胞迁移能力和氧化应激反应,抑制EMT(P<0.05)。结论:Smad4调控万古霉素诱导的HK-2细胞氧化应激反应和EMT。

Objective:To explore the effect of lentivirus-mediated overexpression of Smad4 or silencing of Smad4 on the epithelial-mesenchymal transition(EMT)of renal tubular epithelial cells HK-2 induced by vancomycin.Methods:Design and package Smad4 silenced or overexpressed chronic virus,and then use the obtained lentivirus to transiently transfect HK-2,the HK-2 cells were divided into vancomycin group,empty vector group(transfected with HBLV-GFP-PURO),Smad4 overexpression group(transfected with HBLV-h-Smad4-GFP-PURO),and Smad4 silencing group(transfected with HBLV-h-Smad4-shRNA-GFP-PURO),select normal HK-2 cells from the control group.Cell migration ability,oxidative stress index(TGF-β_(1),SOD,GSH,MDA)in supernatant,protein expression levels of Smad4,E-cadherin,FN,Vimentin were compared in the five groups.Results:Compared with the control group,the migration ability of HK-2 cells,the contents of TGF-β_(1) and MDA in the supernatant,the expression levels of Smad4,FN,Vimentin protein in the vancomycin group and empty vector group were higher(P<0.05),while the contents of SOD and GSH,the expression level of E-cadherin protein were lower(P<0.05).Overexpression of Smad4 increased the migration ability and oxidative stress response of HK-2 cells,and promoted EMT(P<0.05).Silencing Smad4 decreased the migration ability and oxidative stress response of HK-2 cell,and inhibit EMT(P<0.05).Conclusion:Smad4 regulates vancomycin-induced oxidative stress and EMT in HK-2 cells.