Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation.
Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation.
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DOI：
10.3402/jev.v1i0.18397
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发表时间：
2012
影响因子：
16
通讯作者：
Théry C
中科院分区：
文献类型：
--
作者：
Bobrie A;Colombo M;Krumeich S;Raposo G;Théry C
胞外体是直径为50-100 nm的胞外小泡，由多种类型的细胞释放。胞外体是在细胞内的内体隔室中形成的，并在这些隔室与质膜融合时分泌。细胞还分泌其他类型的膜小泡，例如，通过从质膜向外萌发，尽管其中一些在结构特征上明显不同于外体(更大的尺寸)，但其他可能更难分离。在这里，我们使用Rab27a抑制来调节外切体的分泌，我们证明了通过经典的超速离心法从小鼠肿瘤细胞条件培养液中纯化后，至少存在两种不同的囊泡群体。抑制Rab27a可导致一些常规外切体标志物(CD63、Tsg101、Alix和Hsc70)的囊泡分泌减少，但不影响其他标志物(CD9和Mfge8)的分泌。在电子显微镜下，CD9分布在直径从30 nm到150 nm以上的不同大小的囊泡上。在蔗糖梯度上浮选显示CD63、CD9和Mfge8的比例不同，不仅在经典的外切体密度(约1.15g/ml)中，而且在密度超过1.20g/ml的部分中，表明存在异质囊泡种群。CD9和Mfge8也在低速造粒的大小泡中被发现，因此不能被认为是内体来源的小泡的特定成分。我们认为，最常用的外体制剂方案是从内体和其他来源(可能是质膜)共同提纯囊泡。未来的工作将需要改进的技术，以准确纯化和表征不同群体的细胞外小泡。
Exosomes are extracellular vesicles of 50 to 100 nm in diameter, released by many cell types. Exosomes are formed inside the cell in intracellular endosomal compartments and are secreted upon fusion of these compartments with the plasma membrane. Cells also secrete other types of membrane vesicles, for instance, by outward budding from the plasma membrane, and although some of them clearly differ from exosomes by their structural features (larger size), others are possibly more difficult to separate. Here, using Rab27a inhibition to modulate exosome secretion, we show the existence of at least 2 distinct populations of vesicles after purification by classical ultracentrifugation from mouse tumor cell conditioned medium. Rab27a inhibition lead to decreased vesicular secretion of some conventional markers of exosomes (CD63, Tsg101, Alix and Hsc70) but did not affect secretion of others (CD9 and Mfge8). By electron microscopy, CD9 was observed on vesicles of various sizes, ranging from 30 nm to more than 150 nm in diameter. Flotation onto sucrose gradients showed different proportions of CD63, CD9 and Mfge8 not only in fractions of densities classically described for exosomes (around 1.15 g/ml) but also in fractions of densities over 1.20 g/ml, indicating the presence of heterogenous vesicle populations. CD9 and Mfge8 were also found in large vesicles pelleted at low speed and can thus not be considered as specific components of endosome-derived vesicles. We propose that the most commonly used protocols for exosome preparations co-purify vesicles from endosomal and other origins, possibly the plasma membrane. Future work will be required to improve techniques for accurate purification and characterization of the different populations of extracellular vesicles.