细胞外囊泡(EV)由中枢神经系统中的所有细胞分泌,包括神经元和星形胶质细胞。EV是脂质膜包封的颗粒,其装载有反映起源细胞的动态活性的各种生物活性货物。与神经元相反,星形胶质细胞释放的电动汽车的具体作用不太清楚,部分原因是难以将原代星形胶质细胞培养维持在静止状态。本研究的目的是建立一个人无血清星形胶质细胞培养系统,将原代星形胶质细胞维持在静止状态,以研究形态学,函数,和星形胶质细胞衍生的电动汽车的蛋白质货物。将具有G5补充物的无血清培养基和具有2%FBS的血清补充物培养基与市售人类原代胎儿星形胶质细胞的培养进行比较。无血清星形胶质细胞表现出与体内星形胶质细胞相似的形态,令人惊讶的是,与FBS中慢性培养的星形胶质细胞相比,星形胶质细胞标志物水平更高。相比之下,无血清星形胶质细胞中的星形胶质细胞和炎症标志物在急性2%FBS或细胞因子暴露后24小时上调,确认他们的反应能力。重要的是,这提示急性和慢性星形胶质细胞反应性涉及不同的信号通路.尽管形态相似,长期血清培养的星形胶质细胞来源的EV(ADEVs)与无血清的ADEVs相比体积较小,可以重新激活无血清的星形胶质细胞.蛋白质组学分析确定了两种类型ADEVs的不同蛋白质数据集,富含慢性血清培养的星形胶质细胞衍生的EVs的补体和凝血级联,提供他们在中枢神经系统中的角色的见解。总的来说,这些结果表明,在无血清培养基中培养的人原代星形胶质细胞与体内静止的星形胶质细胞具有相似性,血清的加入诱导了人反应性星形胶质细胞及其ADEVs特有的多种形态和转录变化.因此,应该更加强调使用多种结构,分子,和功能参数评估ADEVs作为星形胶质细胞健康的生物标志物。
Extracellular vesicles (EVs) are secreted by all cells in the CNS, including neurons and astrocytes. EVs are lipid membrane enclosed particles loaded with various bioactive cargoes reflecting the dynamic activities of cells of origin. In contrast to neurons, the specific role of EVs released by astrocytes is less well understood, partly due to the difficulty in maintaining primary astrocyte cultures in a quiescent state. The aim of this study was to establish a human serum-free astrocyte culture system that maintains primary astrocytes in a quiescent state to study the morphology, function, and protein cargoes of astrocyte-derived EVs. Serum-free medium with G5 supplement and serum-supplemented medium with 2% FBS were compared for the culture of commercially available human primary fetal astrocytes. Serum-free astrocytes displayed morphologies similar to in vivo astrocytes, and surprisingly, higher levels of astrocyte markers compared to astrocytes chronically cultured in FBS. In contrast, astrocyte and inflammatory markers in serum-free astrocytes were upregulated 24 h after either acute 2% FBS or cytokine exposure, confirming their capacity to become reactive. Importantly, this suggests that distinct signaling pathways are involved in acute and chronic astrocyte
reactivity. Despite having a similar morphology, chronically serum-cultured astrocyte-derived EVs (ADEVs) were smaller in size compared to serum-free ADEVs and could reactivate serum-free astrocytes. Proteomic analysis identified distinct protein datasets for both types of ADEVs with enrichment of complement and coagulation cascades for chronically serum-cultured astrocyte-derived EVs, offering insights into their roles in the CNS. Collectively, these results suggest that human primary astrocytes cultured in serum-free medium bear similarities with in vivo quiescent astrocytes and the addition of serum induces multiple morphological and transcriptional changes that are specific to human reactive astrocytes and their ADEVs. Thus, more emphasis should be made on using multiple structural, molecular, and functional parameters when evaluating ADEVs as biomarkers of astrocyte health.