Rani Sweta, O'Brien Keith, Kelleher Fergal C, Corcoran Claire, Germano Serena, Radomski Marek W, Crown John, O'Driscoll Lorraine
School of Pharmacy & Pharmaceutical Sciences, Panoz Institute, Trinity College Dublin, Ireland.
Methods Mol Biol. 2011;784:181-95. doi: 10.1007/978-1-61779-289-2_13.
Exosomes are nano-sized, cell membrane surrounded structures that are released from many cell types. These exosomes are believed to transport a range of molecules, including mRNAs, miRNAs, and proteins; the contents depending on their cell of origin. The physiological and pathological relevance of exosomes has yet to be fully elucidated. Exosomes have been implicated in cell-to-cell communication. For example, in relation to the immune system, such exosomes may enable exchange of antigen or major histocompatibility complex-peptide complexes between antigen-bearing cells and antigen-presenting cells; in cancer, they may contain molecules that not only have relevance as biomarkers, but may also be taken up and cause adverse effects on secondary cells. Furthermore, exosomes have been proposed as autologous delivery systems that could be exploited for personalised delivery of therapeutics. In order to explore the contents and functional relevance of exosomes from medium conditioned by culture cells or from other biological fluids, prior to extensive molecular profiling, they must be isolated and purified. Here, we describe differential centrifugation methods suitable for isolating exosomes from conditioned medium and from other biological fluids, including serum, saliva, tumour ascites, and urine. We also detail Western blotting and transmission electron microscopy methods suitable for basic assessment of their presence, size, and purity, prior to progressing to global mRNA, miRNA, or protein profiling.
外泌体是纳米级的、被细胞膜包裹的结构，可从多种细胞类型中释放出来。据信这些外泌体可运输一系列分子，包括信使核糖核酸（mRNA）、微小核糖核酸（miRNA）和蛋白质；其内容物取决于它们的起源细胞。外泌体的生理和病理相关性尚未完全阐明。外泌体与细胞间通讯有关。例如，在免疫系统方面，此类外泌体可能使携带抗原的细胞与抗原呈递细胞之间能够交换抗原或主要组织相容性复合体-肽复合物；在癌症中，它们可能含有不仅与生物标志物相关，而且可能被次级细胞摄取并产生不利影响的分子。此外，外泌体已被提议作为自体递送系统，可用于个性化治疗递送。为了探索来自培养细胞条件培养基或其他生物流体的外泌体的内容物及其功能相关性，在进行广泛的分子分析之前，必须对其进行分离和纯化。在此，我们描述了适用于从条件培养基以及血清、唾液、肿瘤腹水和尿液等其他生物流体中分离外泌体的差速离心方法。我们还详细介绍了蛋白质免疫印迹法和透射电子显微镜法，适用于在进行整体mRNA、miRNA或蛋白质分析之前，对其存在、大小和纯度进行基本评估。
