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目的观察CD2相关蛋白(CD2AP)在肾脏不同细胞系中的表达分布,及其与足细胞裂孔隔膜分子nephrin和细胞骨架蛋白F-actin之间的联系。方法以DMEM培养基培养人肾小球系膜细胞(HMC)和人肾小管上皮细胞系(HK-2),RPMI 1640培养基培养条件永生化小鼠足细胞系。以RT-PCR及Western blotting方法检测足细胞内CD2AP和nephrin的表达。间接免疫荧光结合激光共焦方法观察CD2AP在HMC、HK-2、未分化及已分化足细胞中的表达情况,及CD2AP与nephrin在足细胞中的共存。直接免疫荧光结合激光共焦方法观察F-actin在足细胞的表达及其与CD2AP的共存。结果CD2AP均匀分布于HK-2及未分化足细胞的核周及胞浆,而不表达于HMC细胞。在足细胞分化过程中,CD2AP的分布发生了变化,出现向周边聚集的现象。CD2AP与足细胞裂孔隔膜分子nephrin及细胞骨架蛋白F-actin在足细胞中存在共定位关系。结论CD2AP表达于上皮来源的肾脏固有细胞。CD2AP在足细胞中的分布特点,提示CD2AP可能参与足细胞的分化过程,并与裂孔隔膜分子功能及细胞骨架调节有关。
Objective To observe the expression and distribution of CD2-related protein (CD2AP) in different kidney cell lines and its relationship with the nephrin and cytoskeletal protein F-actin. Methods Human mesangial cells (HMC) and human renal tubular epithelial cell line (HK-2) were cultured in DMEM medium and immortalized mouse podocyte lines were cultured in RPMI 1640 medium. The expressions of CD2AP and nephrin in podocytes were detected by RT-PCR and Western blotting. The expression of CD2AP in HMC, HK-2, undifferentiated and differentiated podocytes and the coexistence of CD2AP and nephrin in podocytes were observed by indirect immunofluorescence combined with confocal laser scanning. The expression of F-actin in podocytes and its coexistence with CD2AP were observed by direct immunofluorescence combined with confocal laser scanning. Results CD2AP was evenly distributed in the perinuclear and cytoplasm of HK-2 and undifferentiated podocytes, but not in HMC cells. During the process of podocyte differentiation, the distribution of CD2AP changes and appears to gather around. CD2AP and podocyte pore membrane molecules nephrin and cytoskeletal protein F-actin colocalization in podocytes. Conclusion CD2AP is expressed in epithelial-derived renal cells. The distribution of CD2AP in podocytes suggests that CD2AP may be involved in the process of podocyte differentiation and is related to the molecular function and the cytoskeleton regulation of hiatus.