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目的:通过调控足细胞Src激酶的表达,观察足细胞表型的改变;并探讨Src激酶在血管紧张素Ⅱ(AngⅡ)诱导的足细胞损伤中的作用。方法:体外培养条件永生化小鼠足细胞(MPCs),并分组如下:A:正常对照组;B:Src激酶干扰RNA(siRNA)组;C:PP2(Src激酶抑制剂)组;D:AngⅡ组;E:AngⅡ+Src激酶siRNA组;F:AngⅡ+PP2组。采用免疫印迹法检测Src激酶及其磷酸化水平;AnnexinⅤ-FITC/PI双染及Hoechst染色法检测细胞凋亡;荧光显微镜观察FITC-鬼笔环肽染色的细胞骨架;划痕实验进行细胞运动性分析。结果:1免疫印迹法示:应用Src激酶siRNA或PP2干预MPCs后,Src激酶表达较A组下降(P<0.05);AngⅡ处理后,MPCs内Src激酶生成与A组相比明显增多(P<0.05);Src激酶siRNA或PP2干预MPCs可减少AngⅡ上调的Src激酶表达(P<0.05)。2 AngⅡ诱导MPCs内Src激酶及其磷酸化形式表达的升高,在一定范围内呈时间及浓度依赖性。3Annexin V-FITC/PI双染法及Hoechst染色法两种方法显示:A组、B组与C组MPCs凋亡无明显差异(P>0.05);AngⅡ干预后,MPCs凋亡较A组增加(P<0.05);Src激酶siRNA或PP2可减轻AngⅡ诱导的MPCs凋亡(P<0.05)。4荧光显微镜观察FITC-鬼笔环肽染色的细胞骨架:A组、B组与C组MPCs的F-actin呈微丝样结构,聚集成束,沿细胞长轴分布,形成应力纤维;D组F-actin发生重组,微丝样结构消失,排列紊乱,F-actin沿胞体外周分布,形成F-actin环;E组及F组较D组骨架重组有所减轻。5B组及C组与A组相比,MPCs运动性降低(P<0.05);D组MPCs运动性较A组增加(P<0.05);E组及F组较D组MPCs运动性降低(P<0.05)。结论:AngⅡ刺激MPCs胞内Src激酶和p-Src(Tyr 416)激酶表达增加,并在一定范围内呈时间及浓度依赖性。AngⅡ诱导MPCs凋亡增加、骨架改变、运动性增强;该过程与AngⅡ引起Src激酶和p-Src(Tyr 416)激酶生成增加有关。降低细胞内Src激酶浓度,可减轻AngⅡ诱导的MPCs损伤。降低对照组MPCs胞内Src激酶浓度,MPCs运动性降低,但未观察到细胞凋亡、细胞骨架改变。
OBJECTIVE: To observe the changes of podocyte phenotype by regulating the expression of Src kinase in podocytes. To investigate the role of Src kinase in podocyte injury induced by angiotensin Ⅱ (AngⅡ). Methods: Immortalized mouse podocytes (MPCs) were cultured in vitro and divided into the following groups: A: normal control group; B: Src kinase interference RNA (siRNA) group; C: PP2 (Src kinase inhibitor) Group; E: AngⅡ + Src kinase siRNA group; F: AngⅡ + PP2 group. The phosphorylation level of Src kinase was detected by Western blotting. Apoptosis was detected by AnnexinⅤ-FITC / PI double staining and Hoechst staining. The cytoskeleton stained with FITC-phalloidin was observed by fluorescence microscopy. analysis. Results: 1 Western blotting showed that the Src kinase expression was decreased compared with group A (P <0.05) after Src kinase siRNA or PP2 intervention was applied to MPCs; Src kinase production in MPCs was significantly increased after AngⅡ treatment (P < 0.05). Interference of MPC with Src kinase or PP2 reduced the expression of Angc Src kinase (P <0.05). 2 AngⅡinduced the increase of Src kinase and its phosphorylated forms in MPCs, which showed a time-and concentration-dependent manner in a certain range. There was no significant difference in apoptosis of MPCs between group A, group B and group C (P> 0.05). The apoptosis of MPCs was increased after AngⅡ intervention compared with that of group A (P <0.05) .3Annexin V-FITC / PI double staining and Hoechst staining showed that: P <0.05). Src kinase siRNA or PP2 could reduce AngⅡ-induced apoptosis of MPCs (P <0.05). FITC-phalloidin-stained cytoskeleton was observed by fluorescence microscopy. F-actin in group A, group B and group C MPCs showed a microfilament-like structure, clustered into bundles and distributed along the long axis of the cells to form stress fibers. Group D F-actin recombination, microfilament-like structure disappeared, arranged disorder, F-actin along the pericycle distribution, forming F-actin ring; E group and F group than the D group skeleton reorganization reduced. The motility of MPCs in group B and group C was lower than that in group A (P <0.05); the motility of MPCs in group D was higher than that of group A (P <0.05); the motility of MPCs in group E and F was lower than that of group D <0.05). CONCLUSION: The intracellular expression of Src kinase and p-Src (Tyr 416) kinases stimulated by AngⅡ increased in a time-and concentration-dependent manner. AngⅡinduced increased apoptosis, altered skeletal structure and increased motility of MPCs, which was related to the increase of Src kinase and p-Src (Tyr 416) kinase induced by AngⅡ. Decreasing intracellular Src kinase concentration can reduce Ang Ⅱ-induced MPCs injury. The control group MPCs intracellular Src kinase concentration, MPCs motility decreased, but no apoptosis, cytoskeleton changes.