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首页> 外文期刊>Journal of Extracellular Vesicles >Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations
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Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations

机译：碘克沙醇密度梯度超速离心分离人唾液细胞外囊泡及其表征

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Diagnostic methods that focus on the extracellular vesicles (EVs) present in saliva have been attracting great attention because of their non-invasiveness. EVs contain biomolecules such as proteins, messenger RNA (mRNA) and microRNA (miRNA), which originate from cells that release EVs, making them an ideal source for liquid biopsy. Although there have been many reports on density-based fractionation of EVs from blood and urine, the number of reports on EVs from saliva has been limited, most probably because of the difficulties in separating EVs from viscous saliva using density gradient centrifugation. This article establishes a protocol for the isolation of EVs from human saliva using density gradient centrifugation. The fractionated salivary EVs were characterized by atomic force microscopy, western blot and reverse transcription polymerase chain reaction. The results indicate that salivary EVs have a smaller diameter (47.8±12.3 nm) and higher density (1.11 g/ml) than EVs isolated from conditioned cell media (74.0±23.5 nm and 1.06 g/ml, respectively). Additionally, to improve the throughput of density-based fractionation of EVs, the original protocol was further modified by using a fixed angle rotor instead of a swinging rotor. It was also confirmed that several miRNAs were expressed strongly in the EV-marker-expressing fractions.
机译：专注于唾液中细胞外囊泡（EVs）的诊断方法因其无创性而备受关注。 EV包含蛋白质，信使RNA（mRNA）和microRNA（miRNA）等生物分子，这些分子源自释放EV的细胞，使其成为液体活检的理想来源。尽管有许多关于从血液和尿液中对电动汽车进行基于密度分级的报道，但有关唾液中电动汽车的报道却受到限制，这很可能是因为使用密度梯度离心法很难将电动汽车与粘性唾液分离。本文建立了使用密度梯度离心技术从人唾液中分离电动汽车的方案。通过原子力显微镜，Western印迹和逆转录聚合酶链反应对分离的唾液EV进行了表征。结果表明，与从条件细胞培养基中分离出的电动汽车（分别为74.0±23.5 nm和1.06 g / ml）相比，唾液电动汽车具有更小的直径（47.8±12.3 nm）和更高的密度（1.11 g / ml）。此外，为了提高基于密度的电动汽车的分馏通量，通过使用固定角度转子而不是摆动转子进一步修改了原始协议。还证实了几种miRNA在表达EV标记的部分中强烈表达。

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