摘要
目的细胞外囊泡(extracellular vesicles,EVs)称为细胞间“信使”,通过传递物质和信息调控受体细胞的功能和表型,在生理状态维持及疾病进程中发挥重要作用。然而,亚微米直径、高异质性、低折射率等特性使EVs的分析鉴定严重受阻。本研究旨在探索一种准确、高效的EVs流式检测方案,为EVs的功能研究提供技术支撑。方法首先采用差速离心法分离获得EVs,用已知直径的聚苯乙烯微球作为标尺,对流式细胞仪检测EVs的各项参数进行调试和优化以达到最佳信噪比;梯度稀释EVs样本,优化其上样浓度以降低群检测效应(swarming effect),在此基础上进一步用PKH26和CFSE两种荧光染料对EVs脂膜和蛋白进行双荧光标记,从而实现对EVs物理和生化特性的多参数分析,最后通过特异性抗体标记、电镜观察、纳米颗粒跟踪分析对EVs样本进行验证分析。结果参数优化后的流式细胞仪在散色光通道能将80 nm的微球信号与仪器背景噪音信号清楚分开;通过对样本的梯度稀释建立的浓度与稀释倍数之间的标准工作曲线的相关系数达0.99以上,说明仪器可以对EVs进行定量分析;荧光染色显示EVs呈CFSE及PKH26双阳性,占比约5.46%,与抗体标记的结果相近;电镜和纳米颗粒跟踪分析验证其粒径及群体分布与流式结果一致。结论联合散射光与脂膜荧光染料标记是一种经济、高效的EVs流式检测方案,有利于EVs流式分析的标准化。
Objective Extracellular vesicles(EVs)are known as intercellular“messengers”,they regulate the function and phenotype of recipient cells by delivering substances and messages and play an important role in physiological state maintenance and disease progression.However,the characteristics of EVs,such as submicron diameter,high heterogeneity and low refractive index seriously hinder the identification of these EVs.The purpose of this study is to explore an accurate and efficient flow detection protocol for EVs,and to provide technical support for the functional research of EVs.Methods First,EVs were separated by differential centrifugation,then we adjusted and optimized the flow cytometric detection parameters with the help of polystyrene microspheres of known diameter to achieve the best signal to noise ratio(SNR).Gradient dilution of EVs was performed to reduce swarming effect.On the basis of scattering light,we performed a lipid membrane labeling strategy,the EVs were further double labeled with PKH26 and CFSE,so as to realize the multi-parameters analysis of the physical and biochemical characteristics of EVs.Finally,we verified the flow detection results by specific antibody labeling,electron microscopy and nanoparticle tracking analysis.Results The optimized flow cytometer clearly separated the 80 nm microsphere signal from the background noise signal in the scattering light channel.The correlation coefficient of the established standard working curve between the detected concentration and dilution ratio by gradient dilution was above 0.99,indicating that the instrument could be used for quantitative analysis of EVs.The fluorescence staining showed that the EVs were both positive for CFSE and PKH26,accounting for about 5.46%,which was consistent with the results of antibody labeling.The particle size and population distribution analyzed by electron microscopy and nanoparticle tracking analysis(NTA)were also consistent with the flow results.Conclusion Combining scattered the light and fluorescent labeling o
作者
郭春
李艳伟
王佳佳
邢月婷
黄莹莹
宋兴辉
叶小康
Guo Chun;Li Yanwei;Wang Jiajia;Xing Yueting;Huang Yingying;Song Xinghui;Ye Xiaokang(Core facilities,School of medicine,Zhejiang University,Hangzhou 310058,Zhejiang,China;School of basic medical sciences,Zhejiang University,Hangzhou 310058,Zhejiang,China;Women’s hospital school of medicine,Zhejiang university,Hangzhou 310000,Zhejiang,China)
出处
《中国组织化学与细胞化学杂志》
CAS
CSCD
2023年第3期282-289,共8页
Chinese Journal of Histochemistry and Cytochemistry
基金
教育厅一般科研项目(Y202147028)
浙江大学实验技术研究项目(SYB202130)。
