MECHANISM TO LIMIT CNS BILIRUBIN INFLUX: P-GLYCOPROTEIN

描述(改编自申请人的描述)：非连缀的 高胆红素血症是新生儿最常见的临床症状。 句号。虽然总体上是一种良性的发育现象， 高胆红素血症可能会变得严重，并导致神经损伤 严重的长期神经发育不良后遗症 早期住院的近月儿再次出现核黄体征 出院。高胆红素血症脑病的发病机制尚存 尚不清楚，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，一种依赖于ATP的整合型质膜转运蛋白， 磷酸糖蛋白(P-gp)已被鉴定。P-gp在 脑毛细血管内皮细胞和血管内皮细胞腔内的丰富 限制了大量无关亲脂化合物的脑内流入。 在使用独特的P-gp缺陷零突变转基因的初步研究中 在小鼠品系中，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在脑胆红素内流中的表达 显著增加。这一发现表明P-gp在体内起着重要的作用 在阻止胆红素进入中枢神经系统的作用，这是一个直接的概念 新生儿高胆红素血症脑病发病机制的研究 拟议的实验旨在检验以下两个假设： (1)胆红素是P-gp的底物；(2)胆红素 发育过程中胆红素-P-糖蛋白相互作用与脑内P-糖蛋白表达 受监管的。研究人员建议确定底物的特异性 胆红素与P-gp结合的放射配基研究 正常小鼠和P-gp缺陷小鼠不同发育阶段的脑组织 AGEs；和(Ii)野生型胆红素摄取和外排的体外研究 小鼠脑毛细血管内皮细胞在培养过程中的调节作用 P-gp抑制剂、代谢抑制和抗P-gp单抗 抗体。这些研究将得到体内研究的补充 对照组和P-gp缺失突变小鼠脑内胆红素内流 是否存在P-gp抑制剂的预处理。这个 P-gp在小鼠脑内发育过程中的表达 半定量RT-PCR和Western免疫印迹法。建议的结果 预计研究将检验上述假设。所获取的信息可以 提供关于P-gp在减肥脑中所起作用的新见解 胆红素含量并作为开发模式的推动力 增加新生儿血脑屏障P-gp表达，从而增强保护作用 抗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.