Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells.
Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells.
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DOI：
10.1186/2045-8118-10-4
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发表时间：
2013-01-10
影响因子：
7.3
通讯作者：
Stanimirovic DB
中科院分区：
文献类型：
--
作者：
Haqqani AS;Delaney CE;Tremblay TL;Sodja C;Sandhu JK;Stanimirovic DB
真核细胞除了具有细胞内的囊泡外，还能产生直径为50~1000 nm的胞外微泡，在生理和病理条件下释放或释放到微环境中。这些膜性胞外细胞器包括外体(来源于内体的内部小泡)和外体(来源于质膜的直接萌发/脱落)。细胞外的微囊含有细胞特有的蛋白质、糖蛋白、脂类、核酸和其他分子。这些小泡作为基本的细胞特异性信息的载体，在细胞间的通讯中发挥着重要的作用。大脑中的内皮细胞形成血脑屏障，这是血液和大脑之间的一个专门接口，严格控制两个隔室之间的营养物质和大分子的运输，并与组成神经血管单位的其他细胞密切相互作用。因此，脑内皮细胞胞外微囊可能在病理条件下将脑特异性生物标志物‘外化’到血流中，在血液中的分子跨细胞进入大脑，以及在神经血管单位内的细胞-细胞通讯中发挥重要作用。为了研究脑内皮细胞外切体的细胞特异性分子组成和功能，开发了利用质谱学兼容方法分离细胞外微囊并利用基于质谱学的蛋白质组学表征其特征图谱的方法。从分离的脑内皮细胞胞外微囊中鉴定出1179种蛋白质。通过鉴定近60个已知的标记，包括Alix、TSG101和Tetraspanin蛋白CD81和CD9，验证了这些微囊的有效性。分离的微泡上的表面蛋白可能与原代星形胶质细胞和皮质神经元相互作用，成为细胞间的通讯小泡。最后，脑内皮细胞胞外微囊被证明含有几种先前被证明携带大分子穿过血脑屏障的受体，包括转铁蛋白受体、胰岛素受体、LRPs、低密度脂蛋白和TMEM30A。这里描述的方法允许在不同的生物条件下识别脑内皮细胞特异性细胞外微囊的分子特征。除了是有用生物标志物的潜在来源外，这些囊泡还包含潜在的新型受体，众所周知，这种受体可以通过血脑屏障运送分子。
In addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes) and ectosomes (originating from direct budding/shedding of plasma membranes). Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit. To study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed. A total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially interact with both primary astrocytes and cortical neurons, as cell-cell communication vesicles. Finally, brain endothelial cell extracellular microvesicles were shown to contain several receptors previously shown to carry macromolecules across the blood brain barrier, including transferrin receptor, insulin receptor, LRPs, LDL and TMEM30A. The methods described here permit identification of the molecular signatures for brain endothelial cell-specific extracellular microvesicles under various biological conditions. In addition to being a potential source of useful biomarkers, these vesicles contain potentially novel receptors known for delivering molecules across the blood–brain barrier.