树突状细胞衍生外泌体穿梭微小RNA的分析方法及其在树突状细胞间的水平传播

Methods of analysis of dendritic cell-derived exosome-shuttle microRNA and its horizontal propagation between dendritic cells.

作者信息

Montecalvo Angela, Larregina Adriana T, Morelli Adrian E

机构信息

Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.

出版信息

Methods Mol Biol. 2013;1024:19-40. doi: 10.1007/978-1-62703-453-1_3.

DOI:10.1007/978-1-62703-453-1_3

PMID:23719940

Abstract

Exosomes are extremely small (<100 nm) membrane vesicles, generated in the endocytic compartment that are released to the extracellular milieu by living cells. Although the biological function of exosomes in vivo remains unclear, they seem to function as mechanisms of cell-to-cell communication for horizontal transfer of proteins, antigens, prions, morphogens, mRNA, and noncoding regulatory RNAs, including microRNAs (miRNAs) (also known as exosome-shuttle miRNAs). Dendritic cells (DCs), the most potent professional antigen-presenting leukocytes of the immune system, release relatively high levels of exosomes and also interact with free exosomes present in the extracellular space. Therefore, DCs constitute a good model for the analysis of exosome-shuttle miRNAs and their horizontal propagation between cells. This chapter provides basic protocols for purification of exosomes released by mouse bone marrow-derived DCs, analysis of their miRNA content, and assessment of the function of exosome-shuttle miRNAs, once they are transferred to target/acceptor DCs.

摘要

外泌体是极小的（<100 nm）膜泡，产生于内吞区室，由活细胞释放到细胞外环境中。尽管外泌体在体内的生物学功能尚不清楚，但它们似乎作为细胞间通讯的机制，用于蛋白质、抗原、朊病毒、形态发生素、mRNA和非编码调节RNA（包括微小RNA（miRNA），也称为外泌体穿梭miRNA）的水平转移。树突状细胞（DC）是免疫系统中最强大的专职抗原呈递白细胞，释放相对高水平的外泌体，并且还与细胞外空间中存在的游离外泌体相互作用。因此，DC构成了分析外泌体穿梭miRNA及其在细胞间水平传播的良好模型。本章提供了从鼠骨髓来源的DC释放的外泌体的纯化、其miRNA含量分析以及外泌体穿梭miRNA转移到靶/受体DC后其功能评估的基本方案。