NEONATAL BILIRUBIN NEUROTOXICITY AND P-GLYCOPROTEIN

新生儿胆红素神经毒性和 P-糖蛋白

基本信息

批准号：
6188292
负责人：
JON F WATCHKO
金额：
$ 19.27万
依托单位：
MAGEE-WOMEN'S HOSPITAL OF UPMC
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-08-17 至 2003-07-31
项目状态：
已结题

项目摘要

Unconjugated hyperbilirubinemia is the most common clinical condition in the newborn period and when severe can result in neurologic injury with long term adverse neurodevelopment sequelae as evidenced by the reemergence 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). Recent studies suggest that bilirubin is a substrate for the ATP dependent integral plasma membrane efflux pump phosphoglycoprotein or P-gp. P-gp is expressed in abundance on the luminal aspect of brain capillary endothelial cells and limits the brain influx of a variety of lipophilic compounds. We have observed i) that brain bilirubin uptake is significantly increased in adult P-gp deficient transgenie mice, and ii) that P-gp expression (mRNA and protein) is markedly lower in the fetal and neonatal wild type mouse brain as compared with adults. These findings suggest that P-gp plays an important role in preventing the influx of bilirubin into the CNS and that limited P-gp expression in the newborn brain microvasculature may enhance brain bilirubin uptake in this age group. The proposed experiments are designed to test three hypotheses: 1) that bilirubin interacts with and is transported by P-gp; 2) that brain P-gp expression, levels, and function are regulated in a temporal and spatial fashion, and 3) that metabolic inhibition, hypoxia, and/or a combination of hypoxia/ischemia impairs P-gp function. We will use in vitro (radioligand binding and photoaffinity labeling), cellular (mouse brain capillary endothelial cells, and LLC-PK1 cells transfected with the gene for human or mouse P-gp), and in vivo (wild type and P-gp deficient mice) models to characterize the i) interaction between bilirubin and P-gp, ii) the ontogeny and regional expression of CNS P-gp, and iii) the effects of metabolic inhibition on P-gp. Other than our preliminary studies, there is no information regarding the developmental expression of P-gp in the CNS, and only limited data on the interaction between bilirubin and P-gp and the factors that impact P-gp function. Results of the proposed studies will fully test our above stated hypotheses. The information obtained will provide novel insights regarding the role P-gp plays in attenuating brain bilirubin uptake and serve as an impetus towards developing modalities that increase BBB P-gp expression in newborns thereby enhancing protection against neonatal bilirubin neurotoxicity.

未结合的高胆红素血症是新生儿时期最常见的临床疾病，并且在严重时会导致神经系统损伤，并长期不良神经发育后遗症，这证明了近年婴儿在受早期医院出院的近年婴儿的重复出现。高胆红素性脑病的发病机理尚不清楚，但其发育的核心是胆红素穿过血脑屏障（BBB）进入中枢神经系统（CNS）。最近的研究表明，胆红素是ATP依赖性积分质膜外排泵磷脂糖蛋白或P-gp的底物。 p-gp在脑毛细管内皮细胞的腔内表达，并限制了多种亲脂化合物的脑涌入。我们已经观察到i）I）在成年P-gp缺乏的转基因小鼠中，脑胆红素的摄取显着增加，ii）ii）与成年人相比，胎儿和新生儿野生型小鼠脑中P-gp的表达（mRNA和蛋白质）显着较低。这些发现表明，P-gp在防止胆红素流入中枢神经系统中起着重要作用，并且在新生儿脑微血管系统中的P-gp表达有限可能会增强该年龄段的脑胆红素的吸收。提出的实验旨在检验三个假设：1）胆红素与P-gp相互作用并运输； 2）大脑P-gp表达，水平和功能以时间和空间方式调节，3）代谢抑制，缺氧和/或缺氧/缺血的组合损害了P-gp功能。我们将使用体外（放射素结合和光接触标记），细胞（小鼠脑毛细血管内皮细胞和用人或小鼠P-gp基因转染的LLC-PK1细胞）以及体内（野生型和P-GP缺陷小鼠）在体内和P-gp，p-gp，II和P-gp，II）之间的相互作用来表征II）的模型（野生型和P-GP缺陷）。 iii）代谢抑制对P-gp的影响。除我们的初步研究外，没有有关CNS中P-gp发育表达的信息，只有胆红素和p-gp之间相互作用的数据有限，以及影响p-gp功能的因素。拟议的研究结果将充分检验我们上述假设。获得的信息将提供有关P-gp在减轻脑胆红素摄取的作用的新见解，并成为发展模态的动力，从而增加新生儿中BBB P-gp表达的表达，从而增强了对新生儿胆红素神经毒性的保护。