成分分析

血清外泌体的提取方法与鉴定*

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  • 1.安徽医科大学基础医学院,合肥 230000;
    2.中国人民解放军军事科学院军事医学研究院生命组学研究所,蛋白质药物国家工程研究中心,北京 102206
第一作者 李雪洁 Tel:19955170618; E-mail:catherine980202@163.com
付洁 Tel:13311161618; E-mail:13311161618@163.com
** Tel:13701079575; E-mail:songhf@vip.163.com

收稿日期: 2022-02-28

  网络出版日期: 2024-06-24

基金资助

* 国家自然科学基金面上项目(81272701/H1617)

Extraction method and identification of serum exosomes*

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  • 1. School of Basic Medical Sciences, Anhui Medical University, Hefei 230000, China;
    2. Institute of Lifeomics, Academy of Military Medicine, Chinese Academy of Military Sciences, National Engineering Research Center for Protein Drugs,Beijing 100026, China

Received date: 2022-02-28

  Online published: 2024-06-24

摘要

目的:建立阴选纯化富集(NSPE)法,从血清中高效、快速提取外泌体,并对外泌体进行鉴定。方法:利用低密度脂蛋白受体与低密度脂蛋白(LDL)结合的原理,去除人血清中大量LDL干扰,再通过外泌体表面磷脂分子的磷酸盐基团与二氧化锆(ZrO2)双配位结合的原理富集外泌体。使用纳米流式细胞仪、透射电子显微镜来检测外泌体的粒径分布、颗粒浓度和形态特征,蛋白免疫印迹法检测外泌体表面标志性蛋白。使用基因本体论数据库和京都基因与基因组百科全书数据库对外泌体内部的microRNAs (miRNAs)进行基因通路分析。结果:该方法可在60 min从血清中有效提取外泌体。外泌体颗粒浓度为(2.94±0.3)×1010颗粒·mL-1,粒径大小为(88.07±2.94) nm,并呈现标志性蛋白TSG101+、CD9+。外泌体内部miRNAs与免疫、代谢过程等相关。结论:建立的NSPE法可从血清中高效、快速地提取外泌体,用于健康监测和医学检验。

本文引用格式

李雪洁, 付洁, 冀元凯, 宋海峰 . 血清外泌体的提取方法与鉴定*[J]. 药物分析杂志, 2022 , 42(8) : 1400 -1406 . DOI: 10.16155/j.0254-1793.2022.08.13

Abstract

Objective: To establish a method of negative selection purification-enrichment (NSPE) extract exosomes from serum efficiently and rapidly, and exosomes were identified. Methods: A large amount of low density lipoproteins (LDL) interference in human serum was removed based on the principle of combining the low density lipoprotein receptor with low density lipoprotein. Then, exosomes were enriched by the principle of bidentate binding between the phosphate groups of phospholipid molecules on the surface of exosomes and ZrO2. Nanoflow cytometry and transmission electron microscopy were used to detect the particle size distribution, particle concentration and morphological characteristics of exosomes, and western blotting was used to identify surface specific proteins expression of exosomes. MicroRNAs (miRNAs) in exosomes were analyzed for gene pathway using the Gene Ontology database and the Kyoto Encyclopedia of Genes and Genomes database. Results: The exosomes in serum were effectively extracted using the method here within 60 minutes. The concentration of exosome particles was about (2.94±0.3)×1010 particles·mL-1, and the particle size was (88.07±2.94) nm. The expressions of exosome surface specific proteins, TSG101+ and CD9+, were detected. MiRNAs derived from exosomes were related to immune process and metabolic process. Conclusion: The established NSPE method could be used for rapid and efficient extraction of exosomes from serum for health monitoring and medical examination.

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