CIP4对转化生长因子β1诱导的人肾小管上皮细胞-间充质转分化的影响  

Effect of CIP4 on human renal tubular epithelial to mesenchymal transition induced by transforming growth factor β1

作　　者：白寿军[1] 张亚敏[1] 曾锐[1] 许楚瓯[1] 刘丽丽[1] 周巧丹[1] 李彩霞[1] 裴广畅[1] 葛树旺[1] 徐钢[1] 刘晓城[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院肾内科,武汉430030

出　　处：《中华肾脏病杂志》2011年第4期282-287,共6页Chinese Journal of Nephrology

基　　金：国家自然科学基金（30871172,30971372）;国家自然科学基金青年基金（30800525,30800383）

摘　　要：目的观察骨架调节蛋白CIP4（Cdc42 interacting protein 4）对转化生长因子β1（TGF—β1）诱导的人肾小管上皮细胞-间充质转分化（EMT）的影响，并探讨其产生的机制。方法10μg／L TGF—β1刺激72h诱导人近端肾小管上皮细胞（HK-2细胞）向间充质转分化。Western印迹法检测各组细胞内E—cadherin和α-SMA蛋白的表达。倒置显微镜观察细胞形态的变化。根据GenBank人CIP4的完全cDNA序列，设计1条特异性干扰CIP4表达的RNA片段（CIP4-siRNA）和含野生型CIP4的重组真核表达质粒（pcDNA3．1-hCIP4），利用lipofactamine2000将其转染HK-2细胞。Western印迹法检测对照组、TGF—B1刺激组、CIP4-siRNA转染组、pcDNA3．1-CIP4转染组细胞内CIP4、E—cadhefin和α-SMA蛋白的表达，共聚焦显微镜观察E—cadhefin和仪．SMA蛋白的分布改变；用P13K—Akt特异性抑制剂渥曼青霉素（wortmannin）1panol／L干预TGF—β1刺激的HK-2细胞48h，Western印迹法检测对照组和干预组CIP4表达的变化。结果TGF一[31干预后HK一2细胞E—cadhefin蛋白表达显著减少（P〈0．05），α—SMA蛋白表达显著增多（P〈0．05），细胞形态由典型的上皮细胞向肌成纤维细胞转变，表明TGF-β1诱导。肾小管上皮细胞EMT模型成功。CIP4-siRNA抑制TGF-β1诱导的HK-2细胞表达CIP4后，E—cadhenn蛋白表达显著增多（P〈0．05），α—SMA蛋白表达显著减少（P〈0．05），部分逆转了上述TGF—β1诱导的肾小管上皮细胞EMT。pcDNA3．1-hCIP4转染使CIP4高表达后，HK-2细胞E—cadhefin蛋白表达显著减少（P〈0．05），α-SMA蛋白表达显著增多（P〈0．05），诱导了肾小管上皮细胞EMT。用渥曼青霉素干预TGF—β1刺激的HK-2细胞48h，CIP4可能蛋白表达显著减少（P〈0．05）。结论TGF—β1通过P13K—Akt途径上调CIP4表达，CIP4可能进一步参与TGF—β1诱导的肾小管上皮细胞EMT过程。Objective To observe the effect of CIP4 （Cdc42 interacting protein 4） on human renal tubular epithelial to mesenchymal transition （EMT） induced by transforming growth factor β1 （TGF-β1） and to study the associated mechanism. Methods Human proximal tubular epithelial cells （HK-2 cell line） were cultured with TGF-β1 （10μg/L） for 72 hours. The protein expressions of E-cadherin and α-SMA were measured by Western blotting. One set of siRNA oligos specific for CIP4 and CIP4 construction of the entire coding sequence were designed based on the full CIP4 sequence in GenBank. Then HK-2 cells were transfected with CIP4-siRNA or pcDNA3.1- hCIP4 via lipofactamine 2000. The protein expressions of CIP4, E-cadherin and α-SMA were evaluated respectively in control cells, TGF-β1 treated cells, siRNA transfected cells, pcDNA3.1- hCIP4-transfected cells by Western blotting. The distribution of E-cadherin and α-SMA was observed by confocal microscope. After TGF-β1-treated HK-2 cells were interferenced with specific inhibitor of PI3K-Akt （wortmannin） 1 μmol/L for 48 hours, Western blotting was used to detect the CIP4 protein in control cells and interferenced cells. Results With TGF-β1 stimulation, the expression of E-cadherin protein was decreased markedly （P〈0.05）, and in contract, the expression of α-SMA were increased notably （P〈0.05）, which revealed that TGF-β1 could induce EMT. After transfected with CIP4-siRNA, the protein expression of E-cadherin was increased （P〈0.05）, and the protein expression of α-SMA was decreased （P〈0.05）. The EMT induced by TGF-β1 was effectively reversed. After transfected with pcDNA3.1-hCIP4, the expression of E-cadherin protein was down-regulated （P〈0.05）, and the expression of α-SMA protein was up-regulated compared with control group （P〈0.05）, leading to EMT. After HK-2 cells were interferenced with wortmannin for 48 hours, the expression of CIP4 was decreased （P〈0.05）. Conclusion TGF-β1 upregulates the expression o

关 键 词：转化生长因子Β1 肾小管 上皮细胞 上皮细胞-间充质转分化 CIP4 PI3K-AKT 

分 类 号：R692[医药卫生—泌尿科学]