Proteomic characterisation of leech microglia extracellular vesicles (EVs): comparison between differential ultracentrifugation and Optiprep™ density gradient isolation
Proteomic characterisation of leech microglia extracellular vesicles (EVs): comparison between differential ultracentrifugation and Optiprep™ density gradient isolation
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DOI：
10.1080/20013078.2019.1603048
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发表时间：
2019-01-01
影响因子：
16
通讯作者：
Sautiere, P-E
中科院分区：
文献类型：
--
作者：
Arab, T.;Raffo-Romero, A.;Sautiere, P-E
在哺乳动物中，小胶质细胞被认为是中枢神经系统（CNS）的常驻免疫细胞。许多研究表明，损伤后，这些细胞被激活并在损伤部位募集。水蛭小胶质细胞在实验性中枢神经系统损伤后呈现类似的小胶质细胞活化和迁移模式。这种激活与大量细胞外囊泡（EV）的释放有关。我们收集了由小胶质细胞原代培养物释放的EV，并比较了两种不同的分离方案：一种采用差异超离心（UC），另一种采用额外的Optiprep（TM）密度梯度（ODG）超离心。纳米粒子跟踪分析（NTA）和透射电子显微镜（TEM）用于评估囊泡的大小和形态。通过质谱法评估分离的EV的蛋白质含量。结果表明，在这两个程序中EV特异性蛋白的存在。对每个单一ODG级分的广泛蛋白质组学分析证实了该方案在限制囊泡分离期间共分离的蛋白质聚集体和其他膜颗粒的存在方面的效率。本研究首次允许在环节动物模型中表征小胶质细胞EV蛋白含量。有趣的是，水蛭囊泡中发现的大量蛋白质先前在EV特异性数据库中进行了描述。最后，评估纯化的EV的神经营养活性并促进原代培养的神经元上的神经突生长。
In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep (TM) Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.