Membrane Proteomics of the Blood-Brain Barrier

脑微血管系统由一类特殊的内皮细胞组成，形成细胞屏障。 在血流和大脑间隙之间。这种所谓的血脑屏障(BBB) 区分脑微血管和外周血管床，因为它构成了一个物理和 代谢屏障，严格控制大脑对离子、小分子、蛋白质和循环细胞的摄取。 由于脑内皮细胞质膜同时接触血液和脑间质液，因此它们 它们非常适合作为信号、免疫调节和运输的控制接口 在血液和大脑之间。因此，血脑屏障内皮细胞的许多独特特征可以 可能归因于其质膜的蛋白质组成。全差分等离子体 膜蛋白图谱对于鉴定这些表型决定因素是必要的，但技术上 局限性限制了对这些相对难溶的蛋白质的全球分析。到目前为止，对 血脑屏障膜蛋白含量缺乏，我们对这种动态界面的理解继续是 受有限数量的已知生理和生化属性支配。因此，一部小说 消减抗体表达克隆方法将用于鉴定差异表达的血脑屏障 质膜蛋白与那些在高度血管化的肝、肾、 肺和心脏组织。这种基于抗体的方法将通过使用组合的人单抗来扩展。 链抗体(ScFv)文库用于询问完整脑内皮细胞的质膜表面。 这种互补的方法将导致血脑屏障特异性质膜的同时鉴定 蛋白质和同源单链抗体靶向试剂。已鉴定的血脑屏障特异性血浆的组织分布 将对膜蛋白进行评估，以确定质膜中的数量差异 存在于血脑屏障和上述外周血管床之间的蛋白质表达。这个 差异血脑屏障膜蛋白质组的编辑将有助于阐明血脑屏障紧密的独特方面 连接组成、血脑屏障分子运输网络和血脑屏障参与疾病的能力 发病机制。最后，鉴定的BBB特异性质膜蛋白-单链抗体对将被研究 它们作为非侵入性药物输送渠道的潜力。

The brain microvasculature is comprised of a specialized class of endothelium that forms a cellular barrier between the bloodstream and the interstices of the brain. This so-called blood-brain barrier (BBB) distinguishes the brain microvasculature from peripheral vascular beds because it constitutes a physical and metabolic barrier that tightly regulates brain uptake of ions, small molecules, proteins, and circulating cells. Since brain endothelial cell plasma membranes contact both the bloodstream and brain interstitial fluid, they are ideally positioned to act as the controlling interfaces for signaling, immune regulation, and transport between the blood and brain. Therefore, many of the unique characteristics of the BBB endothelium can likely be attributed to the protein composition of its plasma membranes. Comprehensive differential plasma membrane protein profiling is necessary to identify these phenotypic determinants, but technological limitations have restricted the global analysis of these comparatively insoluble proteins. To date, studies of BBB membrane protein content are lacking and our understanding of this dynamic interface continues to be governed by a limited number of known physiologic and biochemical attributes. Therefore a novel methodology, subtractive antibody expression cloning, will be used to identify differentially expressed BBB plasma membrane proteins compared with those that are expressed in the highly vascularized liver, kidney, lung, and heart tissues. This antibody-based method will be expanded by using combinatorial human single- chain antibody (scFv) libraries to interrogate the plasma membrane surface of intact brain endothelial cells. This complementary method will result in the simultaneous identification of BBB-specific plasma membrane proteins and cognate scFv targeting reagents. The tissue distributions of the identified BBB-specific plasma membrane proteins will be evaluated in order to determine the quantitative differences in plasma membrane protein expression that exist between the BBB and the aforementioned peripheral vascular beds. The compilation of the differential BBB membrane proteome will help elucidate unique aspects of the BBB tight junction composition, the BBB molecular transport network, and the BBB capacity for participation in disease pathogenesis. Finally, identified BBB-specific plasma membrane protein-scFv pairs will be investigated for their potential as noninvasive drug delivery conduits.