Isolation of Human Brain Endothelial Cells and Characterization of Lipid Raft-Associated Proteins by Mass Spectroscopy
Isolation of Human Brain Endothelial Cells and Characterization of Lipid Raft-Associated Proteins by Mass Spectroscopy
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DOI：
10.1007/978-1-60761-938-3_13
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发表时间：
2011-01-01
期刊：
影响因子：
--
通讯作者：
Prat, Alexandre
中科院分区：
文献类型：
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作者：
Cayrol, Romain;Haqqani, Arsalan S.;Prat, Alexandre
血脑屏障(BBB)限制分子、营养物质和细胞从全身血液循环进入中枢神经系统(CNS)，反之亦然，从而为中枢神经系统的发育和功能提供了最佳的微环境。脑内皮细胞(BECs)是血液和中枢神经系统之间的主要屏障。此外，构成神经血管单位的周细胞、神经元和星形胶质细胞支持BEC的功能，对维持这种限制性通透性表型是必不可少的。为了更好地了解BBB特性背后的分子机制，我们提出了一种方法来研究人BEC原代培养中耐洗涤剂微区的蛋白质组，即脂筏。本章介绍了稳健的人脐静脉内皮细胞分离方案、标准组织培养方案、内皮细胞纯度评估方案、脂筏微域分离方法，以及表征膜微域蛋白质含量的质谱分析技术。
The blood-brain barrier (BBB) limits the movements of molecules, nutrients, and cells from the systemic blood circulation into the central nervous system (CNS), and vice versa, thus allowing an optimal microenvironment for CNS development and function. The brain endothelial cells (BECs) form the primary barrier between the blood and the CNS. In addition, pericytes, neurons, and astrocytes that make up the neurovascular unit support the BEC functions and are essential to maintain this restrictive permeability phenotype. To better understand the molecular mechanisms underlying BBB properties, we propose a method to study the proteome of detergent resistant microdomain, namely lipid rafts, from human primary cultures of BECs. This chapter describes a robust human BECs isolation protocol, standard tissue culture protocols, ECs purity assessment protocols, lipid raft microdomain isolation method, and a mass spectrometry analysis technique to characterize the protein content of membrane microdomains.