Comparison of exosomes purified via ultracentrifugation (UC) and Total Exosome lsolation (TEI) reagent from the serum of Marek's disease virus (MDV)-vaccinated and tumor bearing chickens
Comparison of exosomes purified via ultracentrifugation (UC) and Total Exosome lsolation (TEI) reagent from the serum of Marek's disease virus (MDV)-vaccinated and tumor bearing chickens
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DOI：
10.1016/j.jviromet.2018.10.004
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发表时间：
2019-01-01
影响因子：
3.1
通讯作者：
Parcells, Mark S.
中科院分区：
文献类型：
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作者：
Neerukonda, Sabari Nath;Egan, Nicholas A.;Parcells, Mark S.
细胞外囊泡（EV）是用于指由多种细胞产生并释放到间质空间和体液中的微粒、外来体和凋亡小体的集合术语。血清外泌体可以作为非常宝贵的生物标志物，含有m/miRNA、脂质和蛋白质，指示各种状况。目前对通过不同方法纯化的血清外泌体的生物标志物谱的表征和相互共识的研究有限。本研究通过分析exosome的大小分布、浓度、形态和miRNA表达谱，比较了两种常用的血清exosome纯化方法，即超离心法（UC）和总Exosome分离法（TEI）的优缺点。从马立克氏病病毒（MDV）感染的鸡获得血清，所述鸡或者接种了针对马立克氏病（MD）的疫苗并因此受到保护，或者未接种疫苗并携带MDV诱导的肿瘤。进行纳米颗粒跟踪分析（NTA）和透射电子显微镜（TEM）以分别评价粒度、浓度和形态完整性。我们的结果表明，通过任一程序纯化的颗粒的尺寸分布与外泌体的尺寸分布（30-150 nm）一致。TEI试剂产生更高的产率，并共分离出稍大（类似于180 nm）的额外EV群体。基于来自外来体小RNA的先前高通量测序实验的miRNA表达谱，我们选择了六种细胞和四种MDV 1 miRNA，以验证它们在UC和TEI纯化的外来体中的表达。miRNA表达谱显示两种方法之间的相对相关性，但在丰度上观察到明显差异，TEI纯化的外泌体显示出与UC纯化的外泌体相比更高的miRNA表达，这与更高的产率一致。与从携带肿瘤的鸡获得的那些相比，来自接种疫苗的鸡的TEI纯化的外泌体表现出更高的肿瘤抑制miRNA gga-mir-146 b表达和最低的oncomiR gga-mir-21表达。我们提出gga-mir.146和gga-mir. 21可以分别作为疫苗诱导的保护和MD肿瘤的血清外泌体生物标志物。
Extracellular vesicles (EVs) is a collective term used to refer microparticles, exosomes, and apoptotic bodies produced by a variety of cells and released into interstitial spaces and bodily fluids. Serum exosomes can serve as invaluable biomarkers, containing m/miRNAs, lipids, and proteins, indicative of various conditions. There are currently limited studies on the characterization and mutual consensus of biomarker profiles of serum exosomes purified by different methods. Here we compared the advantages and disadvantages of two commonly used serum exosome purification procedures including ultracentrifugation (UC) and Total Exosome Isolation (TEI) reagent, by analyzing exosome size distribution, concentration, morphology and miRNA expression profiles. Serum was obtained from Marek's disease virus (MDV)-infected chickens that were either vaccinated against Marek's disease (MD), and thus protected, or unvaccinated and bearing MDV-induced tumors. Nanoparticle tracking analysis (NTA) and Transmission Electron Microscopy (TEM) were performed to evaluate particle size, concentration, and morphological integrity, respectively. Our results indicate that the size distribution of particles purified by either procedure is consistent with that of exosomes (30-150 nm). TEI reagent generated higher yields and co-isolated additional EV populations that are slightly larger (similar to 180 nm). Based on the miRNA expression profiles from a previous high throughput sequencing experiment of exosome small RNAs, we selected six cellular and four MDV1 miRNAs, to validate their expression in UC- and TEI-purified exosomes. miRNA expression profiles displayed relative correlation between the two procedures, but distinctive differences were observed in abundance with TEI-purified exosomes showing higher miRNA expression consistent with higher yield than those purified by UC. TEI-purified exosomes from vaccinated chickens exhibited greater expression of tumor suppressor miRNA, gga-mir-146b and least expression of oncomiR, gga-mir-21 compared to those obtained from tumor-bearing chickens. We propose that gga-mir.146 and -21 can serve as serum exosome biomarkers for vaccine-induced protection and MD tumors respectively.