Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.
Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.
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DOI：
10.1080/20013078.2017.1294340
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发表时间：
2017
影响因子：
16
通讯作者：
Rasmussen JT
中科院分区：
文献类型：
--
作者：
Blans K;Hansen MS;Sørensen LV;Hvam ML;Howard KA;Möller A;Wiking L;Larsen LB;Rasmussen JT
研究表明，人类和牛奶中的纳米细胞外囊泡（EV）具有免疫调节特性，可以为婴儿提供有益的健康效果。为了评估牛奶EV可能对健康的影响，必须使用来自其他更丰富的具有良好生物活性特性的牛奶结构的高纯度分离物。此外，温和的分离程序对于降低产生囊泡伪影的风险是重要的，特别是在研究EV亚群时。在这项研究中，我们提出了两种分离方法，完成了三个步骤的基础上，体积排阻色谱法（SEC），有效的和可重复的EV分离从原料奶。该方法不需要任何EV造粒，并且可以应用于人乳和牛乳。我们表明，SEC有效地分离磷脂膜囊泡的主要酪蛋白和乳清蛋白成分在两个不同的酪蛋白减少牛奶馏分，与馏分之一，而不使用ultracentrapgation。与其他相关乳组分（如乳脂球）中EV标记蛋白的最低水平相比，乳EV分离株富含乳粘附素、CD9、CD63和CD81。纳米粒子跟踪分析和电子显微镜揭示了牛奶EV分离株中存在大小不一的囊泡结构。脂质薄层色谱分析表明，EV分离物是缺乏三酰甘油，并提出了磷脂的分布不同于由上皮细胞质膜包围的乳脂球。此外，乳EV级分富含RNA，具有与乳脂球不同的和发散的特征。总的来说，我们的数据支持成功的牛奶EV分离可以在几个步骤中完成，而不使用超离心，因为所提出的基于SEC的分离方法有效地分离了人乳和牛乳中的EV。
Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.