Membrane Proteomics of the Blood-Brain Barrier

描述（由申请人提供）：脑微血管系统由一类特殊的内皮细胞组成，在血流和脑间隙之间形成细胞屏障。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，因为它构成了物理和代谢屏障，其严格调节脑对离子、小分子、蛋白质和循环细胞的摄取。由于脑内皮细胞质膜接触血流和脑间质液，因此它们理想地定位为充当信号传导、免疫调节和血液与脑之间的运输的控制界面。因此，BBB内皮的许多独特特征可能归因于其质膜的蛋白质组成。全面的差异质膜蛋白分析是必要的，以确定这些表型的决定因素，但技术的局限性限制了这些相对不溶性蛋白质的全球分析。到目前为止，缺乏对血脑屏障膜蛋白含量的研究，我们对这种动态界面的理解仍然受到有限数量的已知生理和生化属性的影响。因此，一种新的方法，消减抗体表达克隆，将被用来确定差异表达的血脑屏障质膜蛋白相比，在高度血管化的肝，肾，肺，和心脏组织中表达。这种基于抗体的方法将通过使用组合的人单链抗体（scFv）文库来扩增，以询问完整脑内皮细胞的质膜表面。这种互补方法将导致同时鉴定BBB特异性质膜蛋白和同源scFv靶向试剂。将评价所鉴定的BBB特异性质膜蛋白的组织分布，以确定存在于BBB和上述外周血管床之间的质膜蛋白表达的定量差异。编制的差异血脑屏障膜蛋白质组将有助于阐明独特的方面的血脑屏障紧密连接组成，血脑屏障分子转运网络，和血脑屏障的能力，参与疾病的发病机制。最后，将研究鉴定的BBB特异性质膜蛋白-scFv对作为非侵入性药物递送管道的潜力。

DESCRIPTION (provided by applicant): 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.