MECHANISM TO LIMIT CNS BILIRUBIN INFLUX: P-GLYCOPROTEIN

描述（改编自申请人的描述）：未缀合 高胆红素血症是新生儿中最常见的临床病症 期 虽然这通常是一种良性的发育现象， 高胆红素血症可变得严重并导致神经损伤， 严重的长期不良神经发育后遗症， 早期住院的近足月儿再次出现核黄疸 放电 高胆红素血症性脑病的发病机制仍然是 不清楚，但其发展的核心是胆红素通过 血脑屏障（BBB）进入中枢神经系统（CNS）。 最近，一种ATP依赖的整合质膜转运蛋白， 磷酸糖蛋白（P-gp）已被鉴定。 P-gp表达于 丰富的脑毛细血管内皮细胞的管腔方面， 限制了多种不相关的亲脂性化合物的脑内流。 在使用独特的P-gp缺陷无效突变转基因的初步研究中， 研究人员观察到，在没有大脑的情况下， 毛细血管内皮细胞P-gp表达脑胆红素内流 显著增加。 这一发现意味着P-gp发挥着重要作用 在防止胆红素流入中枢神经系统中的作用，直接的概念， 与新生儿高胆红素血症性脑病发病机制的相关性。 所提出的实验旨在检验以下两个假设： (1)胆红素是P-gp的底物，（2） P-gp与血红蛋白的相互作用和脑内P-gp的表达在发育过程中有一定的相关性。 监管. 研究人员建议确定底物特异性 使用（i）P-gp中胆红素的放射性配体结合研究， 来自不同发育阶段的对照和P-gp缺陷小鼠的脑组织 年龄;和（ii）在体外研究胆红素摄取和流出的野生型 培养的小鼠脑毛细血管内皮细胞， P-gp抑制剂、代谢抑制剂和抗P-gp单克隆抗体 抗体的 这些研究将通过体内研究补充， 对照组和P-gp缺陷无效突变小鼠的脑胆红素内流 存在或不存在P-gp抑制剂预处理。 的 小鼠脑中P-gp的发育表达将使用 半定量RT-PCR和Western免疫印迹。 拟议预算的结果 希望通过研究来验证上述假设。 获得的信息可能 提供了关于P-gp在减弱脑损伤中的作用的新见解， 胆红素含量，并作为发展模式的动力， 增加新生儿BBB P-gp表达，从而增强保护作用 新生儿胆红素神经毒性。

DESCRIPTION (Adapted from the Applicant's Description): Unconjugated hyperbilirubinemia is the most common clinical condition in the newborn period. Although generally a benign developmental phenomenon, hyper-bilirubinemia can become severe and result in neurologic injury with profound long term adverse neurodevelopmental sequelae as evidenced by the reappearance of kernicterus in near term infants subject to early hospital discharge. The pathogenesis of hyperbilirubinemic encephalopathy remains unclear but central to its development is the passage of bilirubin across the blood-brain barrier (BBB) into the central nervous system (CNS). Recently, an ATP dependent integral plasma membrane transporter, phosphoglycoprotein (P-gp) has been identified. P-gp is expressed in abundance on the luminal aspect of brain capillary endothelial cells and limits the brain influx of a wide variety of unrelated lipophilic compounds. In preliminary studies using an unique P-gp deficient null mutant transgenic mouse line, the investigators observed that in the absence of brain capillary endothelial cell P-gp expression brain bilirubin influx is significantly increased. This finding implies that P-gp plays an important role in preventing the influx of bilirubin into the CNS, a concept of direct relevance to the pathogenesis of neonatal hyperbilirubinemic encephalopathy. The proposed experiments are designed to test the following two hypotheses: (1) that bilirubin is a substrate for P-gp and (2) that both the bilirubin-P-gp interaction and brain P-gp expression are developmentally regulated. The investigators propose to determine the substrate specificity of bilirubin for P-gp using (i) radioligand binding studies of bilirubin in brain tissue from control and P-gp deficient mice at different developmental ages; and (ii) in vitro studies of bilirubin uptake and efflux by wild type mouse brain capillary endothelial cells in culture subject to modulation by a P-gp inhibitor, metabolic inhibition, and an anti-P-gp monoclonal antibody. These studies will be complemented by in vivo investigations of brain bilirubin influx in control and P-gp deficient null mutant mice in the presence or absence of pretreatment with a P-gp inhibitor. The developmental expression of P-gp in mouse brain will be determined using semi-quantitative RT-PCR and Western imununoblots. Results of the proposed studies are expected to test above hypotheses. The information obtained may provide novel insights regarding the role P-gp plays in attenuating brain bilirubin content and serve as an impetus towards developing modalities that increase BBB P-gp expression in newborns thereby enhancing protection against neonatal bilirubin neurotoxicity.