标题：巨噬细胞来源外泌体的分离与鉴定

      作者：宋枭，薛逸文，郝静，邓润智    南京大学医学院附属口腔医院 南京市口腔医院副院长办公室，江苏 南京 210008

      卷次： 2022年33卷7期

      【摘要】 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 446 μg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。
      【关键词】 外泌体；巨噬细胞；牙周炎；超速离心
      【中图分类号】 R781.4 【文献标识码】 A 【文章编号】 1003—6350（2022）07—0821—04

Isolation and identification of macrophage-derived exosomes.
SONG Xiao, XUE Yi-wen, HAO Jing, DENG Run-zhi.Vice Principal's Office, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing 210008, Jiangsu,CHINA
【Abstract】 Objective To isolate and identify macrophage-derived exosomes, and provide a reference for cur-rent studies on exosomes extraction, and lay a foundation for subsequent studies on exosomes and periodontal tissue re-generation. Methods The macrophage RAW264.7 was cultured in vitro. The exosomes were extracted by ultracentri-fugation and ultrafiltration, and the total protein of exosomes was determined by BCA kit. The morphological charac-teristics of exosomes were observed by transmission electron microscopy. The particle size and concentration weremeasured by particle analyzer. The expression of exosome positive marker proteins CD9, CD81, TSG101, and Calnex-in was detected by western blotting. Results A total of 446 μg exosomes were extracted from 325 mL macrophageculture 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 la-beled proteins CD9 and TSG101 were expressed in exosomes, but the negative labeled protein Calnexin was not ex-pressed. Conclusion Exosomes derived from macrophage RAW264.7 were successfully isolated in this study, whichhas the general characteristics of exosomes and high extraction content and purity.
      【Key words】 Exosome; Macrophages; Periodontitis; Ultracentrifugation