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目的:观察结缔组织生长因子(CTGF)对体外培养的足细胞整合素连接激酶(ILK)、nephrin和desmin表达以及细胞表面形态的影响,以进一步探讨CTGF介导足细胞损伤的机制。方法:不同浓度rhCTGF(0、10、50 ng/ml)干预足细胞24h,观察rhCTGF对ILK、nephrin及desmin表达影响的浓度效应。以浓度为50 ng/ml的rhCTGF干预足细胞0h、24h、48h,观察rhCTGF对ILK、nephrin及desmin表达影响的时间效应。分别用real timeRT-PCR方法和Western blot方法检测ILK、nephrin及desmin mRNA表达水平和蛋白表达水平。采用扫描电镜技术观察rhCTGF对足细胞表面形态的影响。结果:在基础状态下,足细胞表达其标志蛋白nephrin及少量desmin,10 ng/ml rhCTGF可使desmin表达显著增加(P<0.05),而nephrin表达显著下降(P<0.05),随着rhCTGF浓度增加,desmin表达进一步增加(P<0.05),而nephrin表达进一步减少(P<0.05);rhCTGF刺激24h,足细胞desmin表达上调(P<0.05),而nephrin表达下调(P<0.05),48h,desmin表达进一步增加(P<0.05),而nephrin表达进一步减少(P<0.05)。在基础状态下,足细胞表达一定量的ILK,rhCTGF能上调足细胞ILK表达。扫描电镜显示,对照组足细胞胞体表面相对光滑,微绒毛较少;rhCTGF(50 ng/ml)刺激24h后,足细胞细胞表面形态发生改变,形成大量微绒毛。结论:CTGF能引起足细胞nephrin表达下调,desmin表达上调,促进足细胞发生上皮-间充质转分化;CTGF能引起足细胞微绒毛化。同时,CTGF能诱导足细胞ILK表达增加,推测ILK表达上调可能是足细胞损害机制之一。
OBJECTIVE: To observe the effects of connective tissue growth factor (CTGF) on the expression of podocyte integrin-linked kinase (ILK), nephrin and desmin and the cell surface morphology in vitro so as to further explore the mechanism of CTGF-mediated podocyte injury. Methods: Podocytes were treated with different concentrations of rhCTGF (0, 10, 50 ng / ml) for 24h to observe the effect of rhCTGF on the expression of ILK, nephrin and desmin. The effect of rhCTGF on the expression of ILK, nephrin and desmin was observed after the cells were treated with rhCTGF at a concentration of 50 ng / ml for 0h, 24h and 48h. The expression of ILK, nephrin and desmin mRNA and protein were detected by real time RT-PCR and Western blot respectively. Scanning electron microscopy was used to observe the effect of rhCTGF on the surface morphology of podocyte. Results: Under the basal condition, podocyte expressed nephrin and a small amount of desmin and 10 ng / ml rhCTGF significantly increased the expression of desmin and decreased the expression of nephrin significantly (P <0.05). With the increase of rhCTGF concentration (P <0.05). The expression of desmin was up-regulated (P <0.05), while the expression of nephrin was down-regulated (P <0.05) The expression of desmin further increased (P <0.05), while the expression of nephrin decreased further (P <0.05). In the basal state, podocytes express a certain amount of ILK, and rhCTGF can up-regulate podocyte ILK expression. Scanning electron microscopy showed that the surface of podocytes in the control group was relatively smooth with few microvilli. The surface morphology of podocyte changed after rhCTGF (50 ng / ml) stimulation for 24 h, forming a large number of microvilli. CONCLUSION: CTGF can induce the down-regulation of nephrin expression in podocytes, the up-regulation of desmin expression and the promotion of epithelial-mesenchymal transdifferentiation of podocytes. CTGF can induce podocyte microvilliization. At the same time, CTGF can induce podocyte ILK expression increased, speculated that ILK upregulation may be one of the mechanisms of podocyte damage.