Programa de Estudio y Control de Enfermedades Tropicales (PECET), Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia.
Institute of Biotechnology, University of Granada, Granada, Spain.
Methods Mol Biol. 2021;2369:301-317. doi: 10.1007/978-1-0716-1681-9_16.
Extracellular vesicles (EVs) or exovesicles are a heterogeneous group of small cell-derived membranous structures that carry complex cargoes including lipids, proteins, RNA, and DNA. Emerging evidence suggest that EVs secreted by kinetoplastid parasites play a cardinal role in the pathogenesis of diseases they cause, becoming valuable structures for understanding parasite-host interactions. Moreover, the characterization of EVs molecular cargo may provide a new approach to develop alternative tools for diagnosis and therapy of infectious diseases. EVs have a potential use as biomarkers since it contains a repertoire of DNA species that could be detected at different stages of infection by PCR-based assays. Here, we provide a detailed protocol for the isolation of Trypanosoma cruzi-derived EVs and purification of its DNA cargo for subsequent characterization. The methods described here are transferrable to other medically important parasites that are well adapted to grow in vitro and, therefore, suitable volume of EVs-containing supernatants can be obtained.
细胞外囊泡（EVs）或外囊泡是一组异质性的小细胞衍生的膜结构，携带复杂的货物，包括脂质、蛋白质、RNA 和 DNA。新出现的证据表明，动基体寄生虫分泌的 EVs 在它们引起的疾病发病机制中起着至关重要的作用，成为了解寄生虫-宿主相互作用的有价值的结构。此外，EVs 分子货物的特征分析可能为开发用于诊断和治疗传染病的替代工具提供一种新方法。EVs 具有作为生物标志物的潜力，因为它包含一组 DNA 物种，这些物种可以通过基于 PCR 的检测在感染的不同阶段被检测到。在这里，我们提供了一种从克氏锥虫衍生的 EVs 中分离和纯化其 DNA 货物的详细方案，用于后续的特征分析。这里描述的方法可以转移到其他在体外生长良好的医学上重要的寄生虫上，因此可以获得含有 EVs 的上清液的合适体积。
