Trevisan França de Lima Lucas, Müller Bark Juliana, Rasheduzzaman Mohammad, Weeramange Chameera Ekanayake, Punyadeera Chamindie
Queensland University of Technology, The School of Biomedical Sciences, Saliva and Liquid Biopsy Translational Laboratory, Kelvin Grove, QLD, Australia.
Saliva and Liquid Biopsy Translational Laboratory, Griffith Institute for Drug Discovery (GRIDD) and Menzies Health Institute Queensland (MIHQ), Griffith University, Brisbane, QLD, Australia.
Methods Mol Biol. 2022;2504:101-112. doi: 10.1007/978-1-0716-2341-1_8.
Exosomes are small extracellular vesicles secreted by cells and are known to play a key role in intercellular communication. Several studies have associated exosomes with various roles in tumorigenesis and explored their potential as a source of biomarkers for diagnosis and prognosis in cancer research. Exosomes can be isolated from several body fluids, including those that are noninvasively accessible, such as human saliva. This book chapter provides a step-by-step detailed description of techniques that are used for the isolation, quantification, and characterization of exosomes from saliva. These include ultracentrifugation for the isolation, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot (WB) for characterization of exosomes. The NTA approach explores the Brownian motion and light scattering of particles to predict size and concentration. TEM enables visualization of the exosomes which often present a cup-shaped morphology. Western blot is used to detect commonly expressed exosome-associated proteins. Finally, salivary exosomes isolated using these protocols can further be characterized for downstream analysis according to their cargo (proteins, DNA, RNA, miRNA) and utilized for cancer biomarker discovery.
外泌体是细胞分泌的小细胞外囊泡，已知在细胞间通讯中起关键作用。多项研究已将外泌体与肿瘤发生中的各种作用联系起来，并探索了它们作为癌症研究中诊断和预后生物标志物来源的潜力。外泌体可以从多种体液中分离出来，包括那些非侵入性可获取的体液，如人类唾液。本章详细介绍了从唾液中分离、定量和表征外泌体所使用的技术步骤。这些技术包括用于分离的超速离心、用于外泌体表征的纳米颗粒跟踪分析（NTA）、透射电子显微镜（TEM）和蛋白质免疫印迹（WB）。NTA方法通过探索颗粒的布朗运动和光散射来预测大小和浓度。TEM能够观察到通常呈现杯状形态的外泌体。蛋白质免疫印迹用于检测常见表达的外泌体相关蛋白。最后，使用这些方案分离的唾液外泌体可根据其内含物（蛋白质、DNA、RNA、miRNA）进一步进行表征，用于下游分析，并用于癌症生物标志物的发现。
