摘要
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.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2017年第2期126-131,共6页
Chinese Journal of Nephrology
基金
国家自然科学基金(81270762、81570617)