高表达PTEN抑制TGF-β1诱导的肾小管上皮细胞转分化

Overexpression of PTEN Inhibits Renal Epithelial-mesenchymal Trans-differentiation Induced by TGF-β1

目的研究PTEN高表达对TGF-β1诱导肾小管上皮细胞转分化的抑制及其信号传导机制。方法脂质体介导含人全长PTEN基因或空载体转染人肾小管上皮细胞(HKC细胞)48 h,倒置荧光显微镜检测PTEN转染组及空载体转染组绿色荧光蛋白表达;Western blot检测正常组、PTEN转染组、空载体转染组中PTEN蛋白表达;RT-PCR检测3组中PTEN mRNA表达。然后将实验分为正常组、TGF-β1刺激组(给予TGF-β1刺激)、PTEN+TGF-β1组(PTEN基因转染后给予TGF-β1刺激)、空载体+TGF-β1组(空载体转染后给予TGF-β1刺激),Western blot检测各组细胞E-cad-herin、α-SMA、Akt、p-Akt表达水平。结果PTEN基因及空载体转染HKC细胞48 h后可见明显绿色荧光蛋白表达;PTEN转染组PTEN蛋白及PTEN mRNA表达较正常组及空载体转染组细胞均显著上升(均P<0.05)。在正常组、TGF-β1刺激组、PTEN+TGF-β1组、空载体+TGF-β1组中,TGF-β1刺激组及空载体+TGF-β1组较正常组E-cadherin表达明显下降(均P<0.05),而α-SMA及p-Akt表达显著上升(均P<0.05);PTEN+TGF-β1组较TGF-β1刺激组及空载体+TGF-β1组α-SMA及p-Akt表达明显下降(均P<0.05),而E-cadherin表达上升(均P<0.05);各组细胞Akt表达水平差异不明显(P>0.05)。结论PTEN通过阻止PI3K/Akt通路活化而抑制TGF-β1诱导的肾小管上皮细胞转分化。

Objective To investigate the inhibitory effects of overexpression of PTEN on renal epithelial-mesenchymal trans-differentiation induced by TGF-β1,and the signaling transduction mechanism.Methods HKC cells were transfected with GFP-PTEN via lipofectAMINE2000.The efficiency of transfection was detected by fluorescence microscope.The expression of PTEN protein and mRNA in the transfected cells was detected by Western blot and RT-PCR respectively.The experiment was divided into four groups：normal group,TGF-β1 stimulation group,GFP-PTEN＋TGF-β1 group and empty vector＋TGF-β1 group.The expression of E-cadherin,α-SMA,Akt and p-Akt was detected by Western blot.Results Most cells transfected with GFP-PTEN expressed GFP.The expression of PTEN protein and mRNA was strongly increased when HKC cells were transfected with GFP-PTEN（all P〈0.05）.In both TGF-β1 stimulation group and empty vector＋TGF-β1 group,the expression level of E-cadherin was lower（all P〈0.05）,while that of p-Akt and α-SMA was h多igher than in normal group（both P〈0.05）.The expression level of p-Akt and α-SMA in GFP-PTEN＋TGF-β1 group was lower（both P〈0.05）,while that of E-cadherin was higher than in TGF-β1 stimulation group and empty vector＋TGF-β1 group（both P〈0.05）.The expression of Akt was similar in the four groups.Conclusion Overexpression of PTEN can inhibit renal epithelial-mesenchymal trans-differentiation induced by TGF-β1 through suppressing the activation of PI3K/Akt signal pathway.