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。
The role of protein kinase C (PKC) activation in advanced glycation end products (AGEs)-induced epithelial-mesenchymal transition in renal proximal tubular epithelial cells was in-vestigated. HKC cells were divided into three groups: normal group, AGE-BSA group (100 mg/L AGE-BSA) and AGE-BSA+PKC inhibitor (10 μmol/L chelerythrine chloride) group. PKC activity was measured by PKC assay kit. The expression of Vimentin, and phosphorylated β-catenin was detected by using Western blotting, and the content of TGF-β1 was examined by ELISA method. The intracellular disposition of Vimentin was observed by fluorescence microscopy. As compared with normal group, PKC activity was increased significantly in AGE-BSA group. The expression of Vimentin, phosphorylated β-catenin, and TGF-β1 was enhanced significantly in AGE-BSA group. The expression of Vimentin, phosphorylated β-catenin, and TGF-β1 was significantly blocked by chelerythrine chloride. High expression of Vimentin, phosphorylated β-catenin, and TGF-β1 induced by AGE-BSA may be mediated via the activation of PKC signal transduction pathway.
