MOLECULAR CLONING OF BLOOD/BRAIN BARRIER NUTRIENT TRANSPORTERS

该项目的长期目标是克隆、测序、表达和定位 营养转运体在血-脑和血液中的分布- 脑脊液屏障。大脑的内皮细胞 毛细血管调节营养物质进入大脑的吸收，而 脉络丛上皮细胞清除脑脊液中 很多代谢物和药物。人们对此知之甚少 转运体参与了这些重要的过程。我们将克隆血液- 脑屏障转运体使用两种不同的策略。第一个是 以确定已经从其他组织克隆的转运蛋白是否 在血脑屏障中表达。大脑的候选克隆 毛细血管是肾脏的Na+/肌醇共转运体，自分离以来 脑微血管运输这种重要的磷脂前体，并且 脉络丛的候选者之一是肾脏的Na+/核苷 辅助传送器。Northern和Western印迹、原位杂交和 免疫细胞化学技术将被用来绘制 候选克隆人在血液和大脑的界面。第二个策略是 利用表达克隆技术分离特异性脑毛细血管 脉络丛转运蛋白，如L型氨基酸和Na+- 依赖的脯氨酸转运蛋白。目标是使用以下工具 细胞和分子生物学，以获得对营养的详细了解 代谢物在血液和大脑之间的运输。

The long term goal of this project is to clone, sequence, express and map the distribution of nutrient transporters in the blood-brain and blood- cerebrospinal-fluid barriers. The endothelial cells of the brain capillaries regulate the uptake of nutrients into the brain, and the epithelial cells of the choroid plexus clear the cerebrospinal fluid of many metabolites and drugs. Relatively little is known about the transporters involved in these vital processes. We will clone blood- brain barrier transporters using two different strategies. The first is to determine if the transporters already cloned from other tissues are expressed at the blood-brain-barrier. A candidate clone for brain capillaries is the renal Na+/myo-mositol cotransporter, since isolated brain microvessels transport this important phospholipid precursor, and a candidate for the choroid plexus is the renal Na+/nucleoside cotransporter. Northern and Western blots, in situ hybridization, and immunocytochemical techniques will be used to map the distribution of candidate clones at the blood-brain interface. The second strategy is to use expression cloning techniques to isolate specific brain capillary and choroid plexus transporters, e.g. The L-type amino acid and Na+- dependent proline transporters. The objective is to use the tools of cell and molecular biology to obtain a detailed understanding of nutrient and metabolite transport between the blood and brain.