B7-1在血管紧张素Ⅱ诱导足细胞骨架重排中的作用及其机制探讨

Effects of B7-1 on the cytoskeleton rearrangement in podocytes induced by angiotensin Ⅱ

目的观察共刺激分子B7-1对血管紧张素Ⅱ（AngⅡ）诱导的小鼠足细胞骨架重排的影响，探讨B7-1在足细胞病变中的作用以及可能的分子机制。方法体外培养条件永生性小鼠足细胞（MPC），分组如下：正常对照组、AngⅡ刺激组、CTLA-4（B7-1封闭剂）干预组和CTLA-4干预＋AngⅡ刺激组。转染B7—1RNA干扰片段（siRNA）至分化成熟的足细胞，再予10^-6mmol／LAngⅡ刺激12h，观察沉默B7-1基因对AngⅡ刺激下足细胞骨架改变的影响。用流式细胞术检测足细胞B7-1表达；Western印迹法检测足细胞B7-1、nephrin、p-nephrin分子的表达；异硫氰酸荧光素（FITC）-鬼笔环肽荧光染色法观察足细胞骨架蛋白F—actin表达。结果流式细胞术和Western印迹结果显示，正常对照组足细胞B7—1无表达，AngⅡ刺激组足细胞B7．1表达明显增加，且随AngⅡ刺激浓度增加和时间延长B7—1表达增加（均P〈0．05）。与正常对照组比较，AngⅡ刺激组足细胞nephrin和p-nephrin表达量明显下调（均P〈0．05）。FITC-鬼笔环肽荧光染色结果显示，与AngⅡ刺激组比较，CTLA-4干预＋AngⅡ刺激组细胞骨架重排改善，F—actin重组评分（mCFS）明显降低（P〈0．05），提示封闭B7-1可改善AngⅡ对足细胞细胞骨架的破坏；与AngⅡ刺激组比较，转染B7-1 siRNA＋AngⅡ刺激组细胞骨架重排改善，F-actinmCFS亦明显降低（P〈0．05），提示转染B7-1siRNA亦可改善AngII对足细胞细胞骨架的破坏。结论B7．1参与足细胞骨架重排过程，在足细胞损伤中起重要作用。

Objective To observe the effect of costimulatory molecule B7- 1 on cytoskeleton rearrangement in mouse podoeytes induced by angiotensin ]I （Ang lI ）, and to study the underlying molecular mechanism of B7-1 in the pathological changes of podocytes. Methods All euhivation of conditionally immortalized mouse podoeytes （MPC） in vitro were divided into the following groups： normal control group, CTLA-4 group, Ang 11 group （10-6 mmol/L 12 h, 24 h; 10-s mmol/L 12 h, 24 h） and CTLA - 4 with Ang Ⅱ group. Transfect B7 - 1 RNA interference fragment （siRNA） to the mature podocytes, and then restimulated by Ang 11 （10-6 mmol/L 12 h）, the change of podoeyte cytoskeleton after Ang Ⅱ stimulation were observed. The expression of B7 - 1 in each group was assayed by flow cytometry and Western blotting. The nephrin and p-nephrin protein levels in the four groups were also analyzed by Western blotting. At the same time, the podocyte eytoskeleton distribution as indicated by F-actin was observed by fluorescence microscopy. Results Flow cytometry and Western blotting showed that B7 - 1 was not expressed in the normal control group. Ang Ⅱ showed a concentration and time dependent induction of B7-1 expression in mouse podocytes （P 〈 0.05）. Western blotting indicated that Ang 1I induced B7 - 1 protein expression （P 〈 0.05）. Expression of nephrin and p - nephrin was significantly down-regulated by Ang Ⅱ（P 〈 0.05）. Compared with the normal control group, the expression of podoeyte protein nephrin and p- nephrin in Ang II stimulation group was significantly reduced （P 〈 0.05）. Using FITC phalloidin fluorescence staining showed that CTLA-4＋Ang 11 stimulation group cytoskeleton rearrangement was improved significantly and F-actin recombinant score （mCFS） decreased compared with Ang Ⅱ group （P 〈 0.05）, suggesting that Ang Ⅱ led to the disorder of the podocytes cytoskeleton and the destrnetion of the cytoskeleton of podocytes by Ang Ⅱ could be improved after B7 - 1 blocking. Compared with the Ang Ⅱ stimulation, transfeetion of B7 - 1 siRNA ＋ Ang 11 stimulation group improved F-aetin eytoskeletal rearrangement, and mCFS also decreased significantly （P 〈 0.05）, suggesting that transfeetion of B7 - 1 siRNA might improve the damage of Ang Ⅱ on podoeytes cytoskeleton. Conclusions B7-1 participates in the process of cytoskeleton reconstruction and plays an important role in the pathological changes of podocytes.