PDF(Pubmed)
Abstract：
Small extracellular vesicles (sEV) purified from blood have great potential clinically as biomarkers for systemic disease; however interpretation is complicated by release of sEV ex vivo after blood taking. To quantify the problem and devise ways to minimise it, we characterised sEV in paired serum, plasma and platelet poor plasma (PPP) samples from healthy donors. Immunoblotting showed twofold greater abundance of CD9 in sEV fractions from fresh serum than from fresh plasma or PPP. MACSPlex confirmed this, and showed that proteins expressed on platelet sEV, either exclusively (CD41b, CD42a and CD62P) or more widely (HLA-ABC, CD24, CD29 and CD31) were also twofold more abundant; by contrast non-platelet proteins (including CD81) were no different. Storage of plasma (but not serum) increased abundance of platelet and selected leukocyte sEV proteins to at least that of serum, and this could be recapitulated by activating cells in fresh plasma by Ca2+, an effect abrogated in PPP. This suggests that a substantial proportion of sEV in serum and stored plasma were generated ex vivo, which is not the case for fresh plasma or PPP. Thus we provide strategies to minimise ex vivo sEV generation and criteria for identifying those that were present in vivo.
摘要：
从血液中纯化的小细胞外囊泡（sEV）在临床上具有作为全身性疾病生物标志物的巨大潜力；然而，取血后离体释放sEV的解释变得复杂。为了量化问题并设计将其最小化的方法，我们对配对血清中的sEV进行了表征，来自健康供体的血浆和低血小板血浆(PPP)样品。免疫印迹显示来自新鲜血清的sEV级分中CD9的丰度比来自新鲜血浆或PPP的高两倍。MACSPlex证实了这一点，并显示血小板sEV上表达的蛋白质，要么独家(CD41b，CD42a和CD62P)或更广泛的(HLA-ABC，CD24，CD29和CD31）也是两倍丰富；相比之下，非血小板蛋白（包括CD81）没有差异。血浆（而不是血清）的储存增加了血小板和选定的白细胞sEV蛋白的丰度，至少是血清的丰度，这可以通过Ca2+激活新鲜血浆中的细胞来概括，影响在购买力平价中被废除。这表明血清和储存的血浆中相当大比例的sEV是离体产生的，新鲜血浆或PPP并非如此。因此,我们提供了最小化离体sEV产生的策略和用于鉴定存在于体内的那些的标准。
