PI3-K的激活在TGF-β_1诱导肾小管上皮细胞间质转分化过程中的作用

Effect of activated PI3-K on epithelial-mesenchymal trans-differentiation of HK cells induced by TGF-β_1 in vitro

目的探讨磷脂酰肌醇3激酶(PI3K)的激活在转化生长因子β1(TGF-β1)体外诱导人肾小管上皮细胞(HKCs)间质转分化(EMT)过程中的作用及意义。方法将HKCs细胞株分为正常对照组(C)、不同浓度TGF-β1组、TGF-β1+PI3K抑制剂Wort-mannin组。选取不同时相,采用免疫印迹法(Westernblot)测定总Akt(t-Akt)、磷酸化Akt(p-Akt)、α平滑肌肌动蛋白(αSMA)和E钙黏素(E-cadherin)蛋白水平的表达变化;Akt磷酸化水平用磷酸化Akt与总Akt水平的比值表示。结果正常体外培养的HKCs经10ng/mlTGFβ1刺激1h后p-Akt水平显著升高;αSMA蛋白表达水平在10ng/mlTGFβ1诱导48h后表达显著升高,而Ecadherin蛋白表达水平在TGFβ1诱导48h后表达显著下降。同时加用Wortmannin10nmol/L组,p-Akt、αSMA表达明显抑制,E-cadherin表达显著升高。结论PI3K在TGFβ1诱导的EMT过程中被激活,并且参与TGFβ1诱导的EMT过程,特异性阻断PI3K的激活可有效抑制TGFβ1介导的HKCs的EMT过程。

Objective To investigate the effect of activated PI3-K on epithelial-mesenchymal transdifferentiation of HKCs induced by transforming growth factorβ1, （TGF-β1）. Methods The human kidney proximal tubular epithelial cells （HKCs） cultured on plastic plates were divided into following groups： cultured with free serum medium （FSM）; culture in the different concentrations of TGF-β1; cultured in the presence of recombinant human TGF-β1 and PI3-K inhibitor Wortmannin. The expression of total and phosphor-Akt （t-Akt and p- Akt） was assessed at different time points by Western blot,α sooth muscle actin（α-SMA） and E-cadherin were detected by Western blot. Results A marked increase in p-Akt was seen in HKC at lh after being induced by TGF-β1, and its protein level was enhanced in a TGF- β1 concentration-dependent manner. Protein level of α-SMA was increased markedly at 48 hours after the treatment of TGF-β1 , but protein level of E-cadherin was decreased 48 hours after treatment of TGF-β1. Addition of PI3-K inhibitor Wortmannin （10nmol/L） largely abrogated the effect of TGF-~. Wortmannin was showed to dowrvregulate α-SMA and p-Akt expression and in response to TGF-β1 and upregulate E-cadherin expression. Condusion PI3-K was activated in epithelial-mesenchymal trans-differentiation of HKCs promoted by TGF-β1, and PI3-K inhibitor Wortmannin can significantly inhibit TGF-β1, induction of EMT.