Nephrin和Podocin的细胞内共定位及相互作用  

Expression of Nephrin and Podocin in Cells and Interaction between Them in vitro and in vivo

作　　者：秦晓松[1] 谭明旗[2] 塚口裕康 

机构地区：[1]中国医科大学附属盛京医院检验科,沈阳110004 [2]中国医科大学附属盛京医院第二呼吸内科,沈阳110004 [3]德岛大学研究生院生物科学研究所,日本德岛770026

出　　处：《实用儿科临床杂志》2010年第17期1305-1307,1326,共4页Journal of Applied Clinical Pediatrics

基　　金：辽宁省自然科学基金(20082095);辽宁省高校科研计划项目(L20100610)

摘　　要：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 study the cellular distribution and interaction of Nephrin and Podocin.Methods Plasmids containing Nephrin and Podocin were constructed and transfected to COS-7 cells.Double immunofluorescence was performed to detect Nephrin and Podocin mole-cules plasma expressing.A laser scanning confocal microscopy was applied to study the distributions pattern of Nephrin and Podocin.Gluta-thione-S-transferase（GST）-Nephrin and GST-Podocin fusion protein were expressed in competent cell.Co-immunoprecipitation and GST pull-down assay were performed to study the interaction of Nephrin and Podocin in vitro and in vivo.Results Nephrin and Podocin colocallized in COS-7 cell.A substantial portion of Nephrin and Podocin was colocalized on the intracellular vesicle-like structures as well as the edges.Nephrin and Podocin interact with each other via C-terminals in vitro and in vivo.The interaction sites were C-terminals of each other,in detail,1 134-1 241 amino acids of Nephrin and 262-383 amino acids of Podocin interacted in the cytoplasma.Conclusion Physically interaction of Nephrin and Podocin may play key roles in maintaining the structural and functional integration of slit diaphgram.

关 键 词：NEPHRIN PODOCIN 免疫共沉淀 谷胱甘肽S-转移酶pull-down 

分 类 号：R726.9[医药卫生—儿科]