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目的观察β-连环素(catenin)在TGF-β1诱导的上皮细胞-间充质细胞转分化(EMT)过程中对E-钙黏蛋白(cadherin)表达的调控作用。方法应用穿透性多肽技术构建表达底物模仿多肽和对照多肽。以体外培养的人肾小管上皮细胞HK2为研究对象,模型组予10 ng／ml TGF-β1刺激肾小管上皮细胞72 h,底物模仿多肽组和对照多肽组同时分别予2μmol／L的底物模仿多肽或对照多肽干预TGF-β1刺激肾小管上皮细胞:应用Western印迹方法检测各组E-cadherin、α-SMA蛋白的表达及各组β-catenin酪氨酸磷酸化水平。应用激光共聚焦显微镜观察各组细胞β-catenin的分布。结果正常对照组表达E-cadherin,几乎不表达α-SMA,β-catenin酪氨酸磷酸化水平低,主要表达于细胞连接间的细胞膜上。模型组几乎不表达E-cadherin,α-SMA的表达上调,β-catenin酪氨酸磷酸化水平上调,主要表达于细胞核内。底物模仿多肽组与正常组的结果相似;对照多肽组与模型组结果相似。结论人肾小管上皮细胞β-catenin酪氨酸磷酸化水平的上调,使β-catenin向核转移,E-cadherin／β-catenin复合体解离,E-cadherin功能丧失,表达α-SMA,参与TGF-β1诱导的EMT。
Objective To investigate the regulatory effect of β-catenin on the expression of E-cadherin in TGF-β1-induced epithelial-mesenchymal transition (EMT). Methods The penetrating polypeptide technology was used to construct expression mimics and control polypeptides. Human renal tubular epithelial cells HK2 were cultured in vitro. The model group was stimulated with 10 ng / ml TGF-β1 for 72 h, while the substrate-mimicking polypeptide group and the control polypeptide group were respectively treated with 2 μmol / L substrate The mimic polypeptide or control peptide interfered TGF-β1 to stimulate the renal tubular epithelial cells. The expression of E-cadherin and α-SMA protein and the level of β-catenin tyrosine phosphorylation in each group were detected by Western blotting. The distribution of β-catenin in each group of cells was observed by laser confocal microscopy. Results E-cadherin was expressed in normal control group, α-SMA was scarcely expressed, and tyrosine phosphorylation level of β-catenin was low, which was mainly expressed in the cell membrane between cell junctions. E-cadherin was almost not expressed in the model group, and the expression of α-SMA was up-regulated. The level of tyrosine phosphorylation of β-catenin was up-regulated, which was mainly expressed in the nucleus. Substrate mimic polypeptide group and normal group similar results; control peptide group and the model group similar results. Conclusion The up-regulation of tyrosine phosphorylation of β-catenin in human renal tubular epithelial cells leads to nuclear transfer of β-catenin, dissociation of E-cadherin / β-catenin complex, loss of E-cadherin, expression of α-SMA, TGF-β1-induced EMT.