敲低CD2相关蛋白的表达对足细胞黏附和胞质伸展功能的影响

CD2AP depletion impairs the adhesion and spreading function of podocyte

目的观察敲低足细胞CD2相关蛋白（CD2AP）表达对细胞黏附和胞质伸展功能的影响，并探讨其机制。方法用RPMI1640培养基33℃下培养小鼠未分化足细胞系，转染针对CD2AP的小分子干扰RNA（siRNA），设无特异靶位点的scrambing序列即control siRNA转染组作对照。48h后将转染的足细胞制备成单细胞悬液，接种于预铺有Ⅳ型胶原蛋白的96孔板内，33℃下培养90rain后检测足细胞的贴壁率和细胞的伸展面积；流式细胞仪检测抑制CD2AP后足细胞的凋亡率以及在不同氨基核苷嘌呤霉素（PAN）刺激下足细胞的凋亡率；激光共聚焦显微镜下检测F肌动蛋白（F—actin）的分布变化；Western印迹和免疫共沉淀检测nephrin蛋白的表达及其磷酸化水平。结果转染CD2APsiRNA足细胞的黏附率为41．72％±6．07％，显著低于对照组64．46％±8．53％（P〈0．05）；细胞伸展的面积〉200Ixm。的比例（55．86％）亦显著低于对照组（73．61％）。转染CD2APsiRNA后48h足细胞的凋亡率高于对照组[（5．73±0．61）％比（3．26±0．45）％，P〈0．05）。100mg／L的PAN能明显诱导足细胞的凋亡，减少足细胞的黏附率（P〈0．05）。敲低CD2AP的表达后足细胞F—actin的分布发生明显的变化，nephrin蛋白表达和磷酸化水平下降（P〈0．05）。结论敲低CD2AP的表达使足细胞易于凋亡，影响细胞的黏附功能。足细胞骨架蛋白的紊乱和nephrin信号通路的抑制可能是足细胞黏附和伸展功能下降的机制。

Objective To study the effects of CD2-associated protein （CD2AP） on podocyte adhesion and extension ability and to explore its possible mechanism. Methods Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium at 33℃ permissive conditions. The podocytes were transfected with CD2AP small interfering RNA （siRNA） and scrambing sequences labeled with fluorescein were taken as control. The transfected podocytes were trypsinized and seed into collagen Ⅳ coated plates. The relative cell adhesion and cell area were examined 90 rain later. Apoptotic rates of CD2AP siRNA transfected podocytes and different PAN concentrations incubated podocytes were detected by flow cytometer. The distribution of F-actin was observed under laser scanning confocal microscope. Nephrin protein expression and its phosphorylation level were examined by immunofluorescence and Western blot. Results The relative cell adhesion of CD2AP siRNA transfected podocytes was apparently lower than that of control group[（41.72±6.07）% vs （64.46±8.53）%, P〈0.05]. The cell area analysis had the similar result. The apoptotic rate of CD2AP siRNA transfected podocytes was significantly higher than that of the controls [（5.73±0.61）% vs （3.26±0.45）%, P〈O.05]. 100 mg/L PAN could markedly induce podocytes to apoptosis and impair cell adhesion ability （P〈O.05）. Nevertheless, no significant difference was found in cell body spreading （P〉0.05）. The distribution of F-actin in CD2AP depletion podocytes was apparently altered. The expression of nephrin protein and its phosphorylation level was conspicuously descended to some degree （P〈0.05）. Conclusions CD2AP depletion facilitates podocyte apoptosis and impairs cell adhesion function. Cytoskeleton confusion and nephrin signaling weakness caused by CD2AP depletion may be partly responsible for the decline of cell adhesion and spreading.