南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (07): 804-.doi: 10.12122/j.issn.1673-4254.2019.07.09

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TGF-β1/ILK/FSP1信号通路在环孢素诱导肾小管上皮细胞转分化中的作用

白志勋，陆静，杨亦彬   

  • 出版日期:2019-07-20 发布日期:2019-07-20

Role of TGF-β1/ILK/FSP1 signaling pathway in cyclosporin A-induced epithelialmesenchymal transition in cultured renal tubular epithelial cells

  • Online:2019-07-20 Published:2019-07-20

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摘要： 目的通过体外特异阻断试验，探讨转化生长因子-β1/整合素连接激酶/成纤维细胞特异蛋白1（TGF-β1/ILK/FSP1）信号通 路在环孢素A致肾小管上皮细胞转分化中的作用。方法构建并鉴定ILK-RNAi慢病毒表达载体ILKshRNA。将NRK52E细 胞培养分为5组，分别为空白对照组；环孢素诱导组：细胞培养液加环孢素A1 mg/L；TGF-β1干预组：阻断剂（SB431542）10 μmol/L 加环孢素A1 mg/L；ILK干预组：阳性转染ILKshRNA后加入环孢素A1 mg/L；阴性对照组：阴性转染ILKshRNA后加入环孢素 A1 mg/L。实时荧光定量PCR检测TGF-β1、ILK和FSP-1 mRNA的表达，Western blot检测TGF-β1、ILK、FSP-1的蛋白表达。免 疫组化检测细胞爬片α-SMA阳性表达细胞数。结果与空白对照组比较，环孢素诱导组细胞TGF-β1、ILK、FSP-1基因及蛋白表 达量均显著增加（P<0.05）；TGF-β1 干预组经SB431542 处理后，TGF-β1、ILK 和FSP1 水平较环孢素诱导组降低（P<0.05）；经 ILKshRNA沉默后，ILK和FSP1水平较环孢素诱导组降低（P<0.05）。α-SMA阳性细胞数经SB431542和ILKshRNA处理后较 环孢素诱导组降低（P<0.05）。结论TGF-β1/ILK/FSP1信号通路激活是环孢素A诱导大鼠肾小管上皮细胞转分化的重要机制 之一，ILK参与环孢素A诱导大鼠肾小管上皮细胞间充质转分化过程。

Abstract: Objective To explore the role of transforming growth factor-β1/integrin- linked kinase/fibroblast-specific protein 1 (TGF- β1/ILK/FSP1) signaling pathway in cyclosporine A (CsA)-induced renal tubular epithelial cell transdifferentiation. Methods Rat renal tubular epithelial NRK-52E cells were induced with 1 mg/L CsA, treated with TGF-β1 inhibitor (SB431542, 10 μmol/L), or transfected with the ILK-RNAi lentiviral expression vector (ILKshRNA) or a negative control vector before CsA induction. The expressions of TGF-β1, ILK and FSP-1 mRNAs and proteins in the cells were detected using real-time PCR and Western blotting. The positive cells for α-SMA expression were detected by immunohistochemistry. Results Compared with the blank control cells, the cells treated with CsA showed significantly increased levels of TGF-β1, ILK and FSP-1 mRNAs and proteins (P<0.05). The expressions of TGF-β1, ILK and FSP-1 were significantly lower in TGF-β1 inhibitor group than in CsA group (P<0.05). The levels of ILK and FSP-1 were significantly decreased after shRNA-mediated ILK silencing (P<0.05). The number of positive cells for α-SMA was significantly lower in cells treated with SB431542 and in cells with ILK silencing than in the cells treated with CsA alone (P<0.05). Conclusion The activation of TGF-β1/ILK/FSP-1 signaling pathway is an important mechanism for CsA-induced transdifferentiation in rat renal tubular epithelial cells. ILK participates in CsA-induced epithelialmesenchymal transition of renal tubular epithelial cells.