膀胱癌细胞来源外泌体的提取与鉴定

Isolation and Characterization of Exosomes Derived from Bladder Cancer Cells

目的:提取并鉴定膀胱癌5637细胞来源外泌体。方法:收集膀胱癌5637细胞培养上清液,采用多步骤离心法提取膀胱癌5637细胞外泌体。透射电子显微镜观察外泌体形态及颗粒直径。Bradford法定量外泌体蛋白含量。蛋白质免疫印迹鉴定外泌体标志蛋白。结果:20 m L 5637细胞培养基可收集约50-80μg外泌体。膀胱癌5637细胞来源外泌体呈典型的茶杯样形态,外泌体颗粒直径大约在30-150 nm。膀胱癌5637细胞外泌体提取物中可检测到标志蛋白CD63、TSG101、Hsp70和Hsp90表达。结论:多步骤离心法可以用于提取膀胱癌5637细胞外泌体,为后续开展膀胱癌5637细胞外泌体作用与机制的研究奠定了基础。

Objective： To isolate and characterize the exosomes derived from bladder cancer 5637 cells. Methods： Exosomes were isolated from the supernatant of 5637 cells by differential centrifugations. Transmission electron microscopy was used to determine the morphology and size distribution of bladder cancer 5637 cell-derived exosomes. Bradford protein assay was used to measure the protein concentration of exosornes. Western blot analysis was used to determine the exosomal protein markers. Results： A total of 50 to 80 μg exosomes were isolated from 20 mL 5637 cell culture medium. The bladder cancer 5637 cell-derived exosomes showed the typical cup-shaped morphology. The size distribution of exosomes is ranging from 30 to 150 nm in diameter. The exosomal protein markers CD63, TSG 101, Hsp70 and Hsp90 were positively expressed in exosomes isolated from 5637 ceils. Conclusions： The method of differential centrifugations can be used to isolate bladder cancer 5637 cell exosomes which may facilitate further research on the function and mechanism of bladder cancer 5637 cell-derived exosomes.