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为研究nephrin分子在细胞内的转运途径,探讨其在维持肾小球裂孔膜完整性上的作用,构建了nephrin真核表达载体,并转染至COS-7细胞内,应用免疫荧光三重标记的方法,分别进行细胞内及细胞表面的荧光标记,联合笼型蛋白介导的内吞(clathrin-mediated endocytosis,CME)和脂筏介导的内吞(raft-mediated endocytosis,RME)标记物,通过共聚焦显微镜对裂隙膜分子nephrin在不同时间点细胞内的内吞转运特点进行研究。结果发现在转运启动2 min时,68.44%±4.65%的nephrin通过CME途径进行细胞内转运;在20 min时,65.24%±4.02%的nephrin以RME途径进行转运,并且两种转运途径均可被相关途径抑制物所阻断。表明nephrin通过两种途径进行细胞内转运,提示不同的转运途径可能与其实现不同功能有关。
In order to study the intracellular transport of nephrin molecules and its role in maintaining the integrity of the mesangial cell membrane, nephrin eukaryotic expression vector was constructed and transfected into COS-7 cells. The expression of nephrin was detected by immunofluorescence triple labeling Methods: Fluorescent labeling of intracellular and cell surface, combined with clathrin-mediated endocytosis (CME) and raft-mediated endocytosis (RME) Confocal microscopy was used to study the intracellular endocytic transport of nephrin at different time points. As a result, it was found that 68.44% ± 4.65% of nephrin was transported intracellularly via the CME pathway at 2 min after translocation; 65.24% ± 4.02% of nephrin was translocated at RME via 20 min, and both of the transport routes Related pathway inhibitors blocked. Suggesting that nephrin translocates via both pathways, suggesting that different transporter pathways may be involved in their different functions.