Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes.
Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes.
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DOI：
10.3402/jev.v2i0.20677
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发表时间：
2013
影响因子：
16
通讯作者：
Lötvall J
中科院分区：
文献类型：
--
作者：
Crescitelli R;Lässer C;Szabó TG;Kittel A;Eldh M;Dianzani I;Buzás EI;Lötvall J
近年来，旨在了解外泌体以及其他细胞外囊泡生物学的研究数量呈指数级增长。然而，膜囊泡的分类及其分离的适当方案仍在激烈的讨论和调查中。在分离囊泡时，使用能够分离它们的系统至关重要，以避免交叉污染。使用两种基于离心的方案分离从三种不同类型的细胞系：HMC-1、TF-1和BV-2释放的EV。在方案1中，以2，000 × g收集凋亡小体，然后通过0.8 µ m孔过滤上清液，并以12，200 × g沉淀微泡。在方案2中，以16，500 × g将凋亡小体和微泡收集在一起，然后通过0.2 μ m孔过滤上清液，并以120，000 × g沉淀外来体。通过透射电子显微镜、流式细胞术分析细胞外囊泡，并使用Bioanalyzer®研究RNA谱。RNA谱显示，核糖体RNA主要在凋亡小体中检测到，并且在外来体中没有突出的核糖体RNA峰的较小RNA。相比之下，除了从TF-1细胞培养物中收集的微泡外，微泡含有很少或不含RNA。透射电镜观察发现，不同囊泡颗粒的大小、形状和电子密度分布差异很大，具有典型的凋亡小体、微囊泡和外泌体特征。流式细胞术显示在所有研究的囊泡中存在CD63和CD81，以及除了TF-1衍生的囊泡中存在CD9，因为这些细胞不表达CD9。我们的研究结果表明，基于离心的协议是简单而快速的系统来区分细胞外囊泡的亚群。不同的囊泡显示出不同的RNA谱和形态学特征，但使用CD63包被的珠进行流式细胞术分析时，它们是无法区分的。
In recent years, there has been an exponential increase in the number of studies aiming to understand the biology of exosomes, as well as other extracellular vesicles. However, classification of membrane vesicles and the appropriate protocols for their isolation are still under intense discussion and investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid cross-contamination. EVs released from three different kinds of cell lines: HMC-1, TF-1 and BV-2 were isolated using two centrifugation-based protocols. In protocol 1, apoptotic bodies were collected at 2,000×g, followed by filtering the supernatant through 0.8 µm pores and pelleting of microvesicles at 12,200×g. In protocol 2, apoptotic bodies and microvesicles were collected together at 16,500×g, followed by filtering of the supernatant through 0.2 µm pores and pelleting of exosomes at 120,000×g. Extracellular vesicles were analyzed by transmission electron microscopy, flow cytometry and the RNA profiles were investigated using a Bioanalyzer®. RNA profiles showed that ribosomal RNA was primary detectable in apoptotic bodies and smaller RNAs without prominent ribosomal RNA peaks in exosomes. In contrast, microvesicles contained little or no RNA except for microvesicles collected from TF-1 cell cultures. The different vesicle pellets showed highly different distribution of size, shape and electron density with typical apoptotic body, microvesicle and exosome characteristics when analyzed by transmission electron microscopy. Flow cytometry revealed the presence of CD63 and CD81 in all vesicles investigated, as well as CD9 except in the TF-1-derived vesicles, as these cells do not express CD9. Our results demonstrate that centrifugation-based protocols are simple and fast systems to distinguish subpopulations of extracellular vesicles. Different vesicles show different RNA profiles and morphological characteristics, but they are indistinguishable using CD63-coated beads for flow cytometry analysis.