基于Cytoflex流式细胞仪的外泌体表征方法  

Methods in characterization of exosome based Cytoflex Flow Cytometer

作　　者：曹丽 秦棪楠 刘毅杰 李雯[2] 童东东 韩林[2] 王金丽 王晓霏 黄辰[1] Cao Li;Qin Yannan;Liu Yijie;Li Wen;Tong Dongdong;Han Lin;Wang Jinli;Wang Xiaofei;Huang Chen(School of Medicine,Xi'an Jiaotong University,Shaanxi,Xi'an 710061,China;Biomedical Laboratory Center,Xi'an Jiaotong University,Shaanxi,Xi'an 710061,China;Beckman Coulter Commercial Enterprise(China),Shaanxi,Xi'an 710061,China)

机构地区：[1]西安交通大学医学部,西安710061 [2]西安交通大学生物医学实验中心,西安710061 [3]贝克曼库尔特商业公司(中国),西安710061

出　　处：《现代仪器与医疗》2023年第3期39-46,51,共9页Modern Instruments & Medical Treatment

基　　金：国家自然科学基金(32271006)。

摘　　要：目的探讨基于Cytoflex流式细胞仪直接表征外泌体的新方法。方法选择经透射电镜、纳米颗粒追踪、Western blot方法进行常规表征后的外泌体,按照10倍、100倍和1000倍梯度稀释后依托Cytoflex流式细胞仪进行鉴定,并研究不同分离方法、不同超离次数对外泌体特性的影响,同时进一步探究联合标记在外泌体表征的应用可能性。结果Cytoflex流式细胞仪可基于5~10μl外泌体样本检验粒径分布情况、标志物蛋白表征情况(实验样本CD81表征率可达69.79%,CD63表征率为70.55%)以及外泌体相关分子的表达(神经黏附分子CD171表征率为22.74%)。同时,对血清来源外泌体鉴定结果表明发现基于试剂盒的方法分离的血清来源外泌体具有更高的标记率并且富集到更大尺径的颗粒;而不同超离次数的结果启示外泌体粒径大小与超高速离心次数呈负相关关系。结论Cytoflex流式细胞仪表征外泌体具有客观、直接和高效的特点,这一方法的应用也将极大地丰富了外泌体表征的方式,为外泌体相关研究提供新的思路。Objective To explore a new method for directly characterizing exosomes based on Cytoflex flow cytometry.Methods Exosomes routinely characterized by transmission electron microscopy,nanoparticle tracking,and western blot methods were selected,and then identified by the Cytoflex flow cytometry after 10-fold,100-fold,and 1000-fold gradient dilution.Furthermore,the effects of different separation methods and different times of ultracentrifugation on the characteristics of exosomes were also studied,and the possibility of the application of combined labeling in the characterization of exosomes was further explored.Results The Cytoflex flow cytometer can test the particle size distribution,marker protein characterization based on 5~10μl exosome samples(the CD81 characterization rate:69.79%,CD63 characterization rate:70.55%)and exosome-related molecular expression(the expression rate of neural adhesion molecule CD171 was 22.74%).In addition,the identification results of serum-derived exosomes showed that the serum-derived exosomes isolated by the kit-based method had a higher labeling rate and were enriched into larger particle.While the results of different ultracentrifugation times revealed that the particle size of exosomes was negatively correlated with the increasing number of ultracentrifugation times.Conclusion The Cytoflex flow cytometry can characterize exosomes objectively,directly and efficiently.The application of this method will also greatly enrich the way of exosome characterization and may shed a novel light on exosome-related research.

关 键 词：外泌体表征 标志物检测 Cytoflex流式细胞法 

分 类 号：R446.1[医药卫生—诊断学]