一种细胞外囊泡的流式检测方案的探索与建立  

The Exploration and Establishment of a Method to Detect Extracellular Vesicles by Flow Cytometry

作　　者：郭春 王佳佳[1] 李艳伟[1] 邢月婷 宋兴辉[1] 黄莹莹[1] 叶小康 GUO Chun;WANG Jiajia;LI Yanwei;XING Yueting;SONG Xinghui;HUANG Yingying;YE Xiaokang(Core Facilities,School of Medicine,Zhejiang University,Hangzhou 310000,China;School of Basic Medical Sciences,Zhejiang University,Hangzhou 310000,China)

机构地区：[1]浙江大学医学院公共技术平台,杭州310000 [2]浙江大学医学院基础医学系,杭州310000

出　　处：《中国细胞生物学学报》2021年第3期570-577,共8页Chinese Journal of Cell Biology

基　　金：浙江大学实验技术项目(批准号:SJS201913)资助的课题。

摘　　要：该研究旨在探索细胞外囊泡(extracellular vesicles,EVs)分析的标准化流程,建立一种高效的EVs检测方法,为EVs功能及临床转化研究提供技术支撑。实验采用经典的超速离心法分离EVs,借助已知直径的聚苯乙烯微球设定并优化流式检测EVs的参数,联合应用散色光信号及荧光信号对EVs进行双参数分析鉴定,最后通过电镜观察及蛋白质免疫印迹(Western blot,WB)对鉴定结果进行验证。结果显示,参照纳米微球设定的条件可清楚地将100 nm颗粒信号与噪音信号分离,且微球稀释的梯度和检测到的浓度之间呈线性相关,可以对EVs进行定量分析;荧光染色结果显示,EVs呈CFSE(5,6-Carboxyfluorescein diacetate,succinimidyl ester,蛋白结合染料)及FM1-43(fixable analog of FM?1-43 membrane stain,亲脂性染料)双阳性,占比约3%;电镜验证其形态及直径与预期相一致。该研究通过优化流式方案及双荧光参数分析鉴定探索了EVs流式检测的标准化流程,建立了高效、准确的EVs流式检测方法。This study was aimed to explore the standardized process for the analysis of EVs(extracellular vesicles)and establish a highly efficient detection method for EVs,which provided technical support for the function and clinical translational research of EVs.EVs were isolated by classic ultracentrifugation.The flow cytometric detection parameters were setted up and optimized with the help of polystyrene microspheres of known diameter,and then the application of scattered light signal and fluorescence signal were combined to conduct dual-parameter analysis and identification of EVs.Finally the EVs were observed by electron microscope and WB(Western blot)to verify the identification results.The results of the flow cytometry experiment showed that the 100 nm particle signal could be clearly separated from the noise signal with reference to the conditions setted by the nanospheres.There was a good linear correlation between the gradient of the dilution of the microspheres and the detected concentration,which allowed quantitative analysis of EVs.The fluorescent staining results showed that EVs were double positive for CFSE(protein binding dye)and FM1-43(lipophilic dye),accounting for about 3%.Its shape and diameter were verified by electron microscopy and consistent with those reported in the literature.The study explored the standardization process for flow cytometric detection method of EVs through optimized flow cytometric scheme and dual fluorescence parameter analysis and identification,and established an efficient and accurate EVs flow cytometric detection method.

关 键 词：细胞外囊泡 流式细胞术 荧光信号 散射光信号 分析鉴定 

分 类 号：Q2-33[生物学—细胞生物学]