巨噬细胞来源的外泌体不同提取方法的比较.docxVIP

巨噬细胞来源的外泌体不同提取方法的比较 王飘飘;王会会;彭代银;张善堂;陈卫东 【摘要】目的根据聚乙二醇(polyethylene glycol, PEG)沉淀原理,结合超高速离 心改进一种简便、快速的细胞外泌体分离方法.方法采用超高速离心法、PEG6000 沉淀结合超高速离心和EXO Quick试剂盒，分别提取巨噬细胞外泌体.通过透射电 镜、NanoSight ZS及Nanoparticle tracker(NTA)分析外泌体的形态和粒径;蛋白 印迹法鉴定细胞外泌体标志物Alix、TSG101的表达情况.结果PEG6000沉淀结合 超高速离心法获得的外泌体，其粒径分布和差速离心法相当,小于商品化的EXO Quick试剂盒.而外泌体表面蛋白Alix、TSG101在3种方法提取的沉淀物中都有 表达.透射电镜结果显示,3种方法均获得典型的盘状囊泡.结论实验改进的细胞外泌 体提取方法操作简洁、效率高,并且成本较低.％Aim To establish the methods for exosomes isolation from the cell culture medium based on the principle of polyethy-lene glycol (PEG) precipitation combined with ultra- high speed centrifugation. Methods Exosomes were extracted by different centrifugation, PEG6000 precipitation combined with ultra-high speed centrifugation and EXO Quick kit,respectively. The mor-phology and particle size of exosomes were analyzed by transmis-sion electron microscopy,NanoSight ZS and Nano-particle track-er(NTA). Western blot assay was used to identify if the extrac-tion were exosomes. Results According to PEG6000 precipita-tion combined with ultra-high speed centrifugation method, the exosomes were successfully isolated from the culture medium. The morphology and particle size of exosomes,the exosomes iso-lated from PEG6000 precipitation combined with ultra-high speed centrifugation were equal to those of the ultra-high speed centrifugation but smaller to EXO Quick kit. Whats more, the results of transmission electron microscopy showed that the three methods had typical discoid vesicles. Conclusion The im-provement of the exosome isolation from cell culture medium is convenient,easy and cheap. 【期刊名称】《中国药理学通报》 【年(卷)，期】2018(034)004 【总页数】4页(P589-592) 【关键词】巨噬细胞;外泌体;蛋白印迹法;透射电镜;纳米粒子示踪技术;聚乙二醇 【作者】王飘飘;王会会;彭代银;张善堂;陈卫东 【作者单位】安徽中医药大学药学院，安徽合肥230011;安徽中医药大学药学院，安 徽合肥230011;安徽中医药大学药学院，安徽合肥230011;安徽省中药研究与开发 重点实验室，安徽合肥230031;安徽省立医院药剂科，安徽合肥230001;安徽中医 药大学药学院，安徽合肥230011;安徽省中药研究与开发重点实验室，安徽合肥 230031 【正文语种】中文 【中图分类】R329.24;R341;R392-33;R392.11;R977.6 夕卜泌体(exosomes，EXOs)是由大多数细胞分泌的纳米级(30-150 nm)囊泡， 如上皮细胞