BLOOD-BRAIN BARRIER GLUT1 GLUCOSE TRANSPORTER REGULATION

血脑屏障 GLUT1 葡萄糖转运蛋白调节

• 批准号: 6112288
• 负责人: WILLIAM M PARDRIDGE
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6112288
• 关键词: RNA binding protein; affinity chromatography; blood brain barrier; capillary; chimeric proteins; gel mobility shift assay; gene expression; genetic regulatory element; glioma; glucose transport; human tissue; hypoglycemia; immunocytochemistry; laboratory rat; membrane transport proteins; microcirculation; molecular cloning; nucleic acid sequence; posttranscriptional RNA processing; protein structure function; tissue /cell culture; transfection; transport proteins; vascular endothelium

Glucose transport from blood to brain intracellular spaces is limited by transport through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. Recent studies have shown that more than 90% of BBB glucose transport is mediated by the GLUT1 isoform of the sodium independent glucose transporter gene family. The proposed studies will examine role of post-transcriptional mechanisms in regulating BBB GLUT1 mRNA. The role of cytosolic factors that alter GLUT1 mRNA stability in hypoglycemia and in development will be examined. In addition, full-length GLUT1 mRNA will be used in cell-free translation assays to investigate the effects of brain capillary-derived cytosolic proteins in regulating GLUT1 translation efficiency, which prior work has indicated may be impaired at certain periods of development, and in high- grade human glioma cytosolic proteins to identify a novel cis/trans interaction between the GLUT1 3-UTR and a 77 kDa cytosolic protein. The sequence of the GLUT1 3-UTR containing the cis element was localized with RNase T1 mapping experiments, and oligodeoxy-nucleotide competitor studies. The molecular weight of the cytosolic protein interacting with the cis element of the 3'-UTR was identified by ultraviolet (UV) light cross-linking of RNA/cytosol protein complexes. The region spanning the cis element is conserved in GLUT1 of all species, but is not found in other mRNA sequences in GenBank. The identification of this cis/trans- mechanism allows for an examination of its functional role related to either mRNA stability or mRNA translation efficiency. GLUT1 gene expression in brain is confined to the brain capillary endothelium. Therefore, GLUT1, in addition to limiting BBB glucose transport, is also a genetic marker of the differentiation state of the BBB, and the induction of barrier properties within the capillary endothelium by brain-derived factors. Measurement of the induction of GLUT1 mRNA or protein in small amounts of brain capillary endothelial cells grown in primary culture under the influence of brain-derived factors. Measurement of the induction of GLUT1 mRNA or protein in small amounts of brain capillary endothelial cells grown in primary culture under the influence of brain-derived factors is now possible with the recent development of quantitative polymerase chain reaction (QPCR) and ELISA assays. Using a cluster dish format and these assays, it is feasible to fractionate and characterize brain fractions that induce GLUT1 levels. Although transplant models have demonstrated the inductive effects of brain-derived proteins on brain capillary endothelium, to date no brain- derived factors that induce BBB specific genes have been identified.

葡萄糖从血液到脑细胞内的转运受到以下因素的限制 通过大脑毛细血管内皮壁运输，它构成了 体内的血脑屏障(BBB)。最近的研究表明， 90%以上的血脑屏障葡萄糖转运是由GLUT1亚型介导的 属于钠非依赖性葡萄糖转运蛋白基因家族。建议数 研究将检查转录后机制在 调节BBB GLUT1基因的表达。胞浆因子的作用改变 Glut1mRNA在低血糖和发育过程中的稳定性将被检测。 此外，全长GLUT1 mRNA将用于无细胞翻译 脑毛细血管衍生胞浆作用的实验研究 调节GLUT1翻译效率的蛋白质，这是以前的工作 在某些发育阶段可能会受到损害，在高- 分级人脑胶质瘤胞浆蛋白以鉴定一个新的顺式/反式 GLUT1 3-非编码区与77 kDa胞浆蛋白的相互作用这个 含有顺式元件的GLUT1 3-UTR序列用 RNaseT1作图实验和寡核苷酸竞争对手 学习。胞浆蛋白与之相互作用的相对分子质量 用紫外光鉴定了3‘-非编码区的顺式元件 RNA/胞浆蛋白复合体的交联化。这一地区横跨 顺式元件在所有物种的GLUT1中都是保守的，而在 GenBank中的其他mRNA序列。该顺式/反式- 机制允许检查其与以下内容相关的功能角色 无论是信使核糖核酸稳定性还是信使核糖核酸翻译效率。Glut1基因 在脑中的表达仅限于脑内毛细血管内皮细胞。 因此，GLUT1除了限制血脑屏障葡萄糖转运外，还 血脑屏障分化状态的遗传标记，以及 对毛细血管内皮细胞屏障特性的诱导 脑源性因子。GLUT1基因表达水平的测定 微量脑毛细血管内皮细胞生长中的蛋白质 脑源性因素影响下的原代培养。 微量GLUT1基因或蛋白诱导的测定 原代培养的大鼠脑毛细血管内皮细胞 脑源性因素的影响现在可能随着最近的 定量聚合酶链式反应和酶联免疫吸附试验的研究进展 化验。使用集束式培养皿模式和这些检测方法，是可行的 分离和表征可诱导GLUT1水平的脑部分。 尽管移植模型已经证明了 脑毛细血管内皮细胞上的脑源性蛋白，到目前为止还没有脑- 诱导血脑屏障特异性基因的衍生因子已经被确定。