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目的研究肾小球裂孔隔膜分子Nephrin和Podocin的细胞内表达及相互作用。方法构建载有Nephrin和Podocin羧基端的质粒载体,双重转染的COS-7细胞,采用双免疫荧光标记后经共聚焦显微镜观察Nephrin和Podocin的细胞内分布情况;构建载有谷胱甘肽S-转移酶(GST)-Nephrin,-Podocin的质粒载体转化至感受态细菌后提取融合蛋白,应用免疫共沉淀及GST pull-down分析研究体外培养的COS-7细胞内和大鼠肾小球细胞内Nephrin和Podocin蛋白间的相互作用。结果 Nephrin和Podocin在体外培养的COS-7细胞内共存,二者共存于细胞质的囊泡样结构内,尤其在细胞膜上重合为线样结构;体内和体外实验结果均表明二者存在相互作用,具体的作用部位为细胞内段的羧基端,Nephrin 1 134~1 241氨基酸区域与Podocin 262~383氨基酸区域存在相互作用。结论裂孔隔膜分子Nephrin和Podocin具有体内、体外的物理作用,可能与维持裂孔隔膜的功能和结构稳定性有关。
Objective To investigate the intracellular expression and interaction of glomerular cleft membrane molecules Nephrin and Podocin. Methods The plasmid vector carrying the carboxyl terminal of Nephrin and Podocin was constructed and the double transfected COS-7 cells were double-immunofluorescently labeled to observe the intracellular distribution of Nephrin and Podocin by confocal microscopy. Transferase (GST) -Nephrin, -Podocin plasmid vector was transformed into competent bacteria and the fusion protein was extracted, the co-immunoprecipitation and GST pull-down analysis of cultured in vitro and in vivo glomerular mesangial cells Interactions between Nephrin and Podocin proteins. Results Nephrin and Podocin coexisted in vitro in COS-7 cells. Both of them co-existed in the cytoplasm of vesicular-like structures, especially on the cell membrane. The results of both in vivo and in vitro experiments showed that there existed interaction between Nephrin and Podocin, The specific site of action is the carboxy terminal end of the intracellular segment, and the interaction between Nephrin 1 134-1 241 amino acid region and Podocin 262-383 amino acid region. Conclusion Nephrin and Podocin, a pore-forming membrane molecule, have physical and in vitro physical functions, which may be related to the maintenance of the function and structural stability of hiatus.