巨噬细胞来源外泌体的分离与鉴定

Isolation and identification of macrophage-derived exosomes

目的分离并鉴定巨噬细胞来源的外泌体,为当前外泌体的提取研究提供一定参考,并为后续开展外泌体与牙周组织再生的相关研究奠定基础。方法体外培养巨噬细胞RAW264.7,使用超速离心和超滤结合法提取其外泌体,通过BCA试剂盒测定外泌体的蛋白总量,透射电镜下观察其形态特征,粒径分析仪测定其粒径大小及浓度,免疫印迹法检测外泌体阳性标志蛋白CD9、CD81、TSG101、Calnexin的表达。结果325 mL巨噬细胞培养上清液中提取出446μg外泌体,其呈现为内部含有不均匀的低电子密度物质的囊泡状结构,平均粒径约81.20 nm,浓度为(2.90E+10)Particles/mL。阳性标志蛋白CD9、TSG101在外泌体中表达,阴性标志蛋白Calnexin无表达。结论成功分离了巨噬细胞RAW264.7来源的外泌体,具有外泌体的一般特性,提取含量及纯度较高。

Objective To isolate and identify macrophage-derived exosomes,and provide a reference for current studies on exosomes extraction,and lay a foundation for subsequent studies on exosomes and periodontal tissue regeneration.Methods The macrophage RAW264.7 was cultured in vitro.The exosomes were extracted by ultracentrifugation and ultrafiltration,and the total protein of exosomes was determined by BCA kit.The morphological characteristics of exosomes were observed by transmission electron microscopy.The particle size and concentration were measured by particle analyzer.The expression of exosome positive marker proteins CD9,CD81,TSG101,and Calnexin was detected by western blotting.Results A total of 446μg exosomes were extracted from 325 mL macrophage culture supernatant,which presented a vesicular structure containing inhomogeneous low electron density substances,with an average particle size of about 81.20 nm,and the concentration was(2.90E+10)particles/mL.The positive labeled proteins CD9 and TSG101 were expressed in exosomes,but the negative labeled protein Calnexin was not expressed.Conclusion Exosomes derived from macrophage RAW264.7 were successfully isolated in this study,which has the general characteristics of exosomes and high extraction content and purity.