Par-3蛋白在转化生长因子?茁1介导的大鼠肾小管上皮细胞转分化中的作用

目的 观察转化生长因子（TGF)β1诱导的正常大鼠近端肾小管上皮细胞（NRK52E）转分化（EMT）过程中细胞极性蛋白Par-3的表达及上调Par-3蛋白表达对TGF-β1诱导NRK52E细胞转分化进程的影响。 方法 应用TGF-β1 (10 μg/L)刺激NRK52E细胞，采用RT-PCR、Western印迹和免疫荧光方法分别检测E-钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白（α-SMA）、Par-3 mRNA和蛋白的表达；应用Lipofectmine 2000将pKH3-HA-Par-3质粒瞬时转染NEK52E细胞，采用Western印迹观察上调表达Par-3对上述指标的影响。 结果 TGF-β1刺激后，NRK52E细胞α-SMA蛋白和mRNA水平上调，E-cadherin蛋白和mRNA的表达下调；Par-3蛋白表达呈时间依赖模式下调，72 h TGF-β1刺激组与对照组比较，差异有统计学意义（P < 0.05）。但Par-3 mRNA水平在各时间点差异均无统计学意义（P > 0.05）。脂质体转染外源性质粒pKH3-HA-Par-3上调表达Par-3可显著抑制TGF-β1诱导NRK52E细胞α-SMA蛋白的上调表达；逆转E-cadherin蛋白的下调表达。 结论 在TGF-β1诱导NRK52E细胞转分化进程中细胞极性Par-3蛋白表达下调，基因转染上调表达Par-3可部分减轻EMT的程度，提示Par-3蛋白在TGF-β1诱导的肾小管上皮细胞转分化和肾脏纤维化中可发挥保护性作用。

Influence of Par-3 on epithelial-mesenchymal transition of rat proximal tubular epithelial cells

Objective To investigate the expression of Par-3 in epithelial-mesenchymal transition (EMT) of TGF-β1-stimulated NRK52E cells and the effect of over-expression of Par-3 by gene transfection on EMT. Methods NRK52E cells were grown in DMEM medium supplemented by 10% fetal bovine serum. NRK52E cells were cultured in free serum medium for 24 h, then were stimulated by TGF-β1 (10 μg/L). The expression of E-cadherin, α-SMA, Par-3 mRNA and protein were detected by RT-PCR, Western blot and immunofluorescence, respectively. NRK52E cells were transiently transfected with 1 μg pKH3-HA-Par-3 DNA by Lipofectamine 2000, then the expression of E-cadherin， α-SMA and Par-3 protein were detected by Western blot. Empty pKH3 vector was used as negative control. Results The expression of E-cadherin mRNA and protein were markedly decreased in NRK52E cells induced by TGF-β1, and the expression of α-SMA mRNA and protein were dramatically increased. Western blot analysis demonstrated that the protein level of Par-3 was progressively reduced in a time-dependent manner in response to TGF-β1 treatment in NRK52E cells, which was consistent with immunofluorescence staining results. However, TGF-β1 treatment did not affect the mRNA level of Par-3. Forced expression of exogenous Par-3 led to a severe blockage of TGF-β1-induced E-cadherin suppression and α-SMA induction. Conclusion The expression of Par-3 protein is down-regulated in NRK52E cells stimulated by TGF-β1 and overexpression of Par-3 can suppress EMT induced by TGF-β1, which suggests a protective role for Par-3 in TGF-β1-induced tubular EMT and renal fibrosis