Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations.
Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations.
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DOI：
10.3402/jev.v5.30829
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发表时间：
2016
影响因子：
16
通讯作者：
Shiba K
中科院分区：
文献类型：
--
作者：
Iwai K;Minamisawa T;Suga K;Yajima Y;Shiba K
针对唾液中存在的细胞外小泡(EV)的诊断方法因其非侵袭性而引起了人们的极大关注。EV含有蛋白质、信使RNA(MRNA)和微小RNA(MiRNA)等生物分子，它们来自释放EV的细胞，使其成为液体活检的理想来源。虽然已经有很多关于从血液和尿液中分离EVS的报道，但从唾液中分离EVS的报道数量有限，很可能是因为使用密度梯度离心法将EVS从粘性唾液中分离出来是困难的。本文建立了一种用密度梯度离心法从人唾液中分离肠道病毒的方法。用原子力显微镜、免疫印迹和逆转录聚合酶链式反应对分离的唾液EV进行表征。结果表明，唾液EVS的直径(47.8±12.3 nm)和密度(1.11g/ml)分别小于条件培养液中分离的EVS(74.0±23.5 nm和1.06g/ml)。此外，为了提高电动汽车密度分级的吞吐量，对原协议进行了进一步修改，使用了固定角度的转子而不是摆动的转子。也证实了在EV标志物表达组分中有几个miRNAs强表达。
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.