Membrane Proteomics of the Blood-Brain Barrier

脑微血管系统是由一类特殊的内皮细胞构成的细胞屏障 在血液和大脑间隙之间。这种所谓的血脑屏障（BBB） 将脑微血管系统与外周血管床区分开来，因为它构成了一个物理和 代谢屏障，严格调节脑对离子、小分子、蛋白质和循环细胞的摄取。 由于脑内皮细胞质膜与血流和脑间质液接触， 理想地定位为信号传导、免疫调节和运输的控制界面 在血液和大脑之间。因此，血脑屏障内皮的许多独特特征可以 可能是由于其质膜的蛋白质组成。综合鉴别血浆 膜蛋白谱是必要的，以确定这些表型决定因素，但技术 这些局限性限制了对这些相对不溶性蛋白质的全面分析。迄今为止， BBB膜蛋白含量缺乏，我们对这种动态界面的理解仍然是 由有限数量的已知生理和生化属性控制。因此，一部小说 方法学，消减抗体表达克隆，将用于鉴定差异表达的BBB 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克隆抗体来扩展。 链抗体（scFv）文库来询问完整脑内皮细胞的质膜表面。 这种互补的方法将导致BBB特异性质膜的同时鉴定 蛋白和同源scFv靶向试剂。鉴别的BBB特异性血浆的组织分布 将评价膜蛋白，以确定质膜中的定量差异， 存在于BBB和上述外周血管床之间的蛋白质表达。的 差异血脑屏障膜蛋白质组的编译将有助于阐明血脑屏障紧密的独特方面， 连接组成、BBB分子转运网络和BBB参与疾病的能力 发病机制最后，将研究所鉴定的BBB特异性质膜蛋白-scFv对， 它们作为非侵入性药物输送管道的潜力。

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.