Neonatal Bilirubin Neurotoxicity and P-Glycoprotein

新生儿胆红素神经毒性和 P-糖蛋白

• 批准号: 7039022
• 负责人: JON F WATCHKO
• 金额: $ 27.46万
• 依托单位: MAGEE-WOMEN'S RES INST AND FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-17 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7039022
• 关键词: P glycoprotein; apoptosis; bilirubin; biological transport; blood brain barrier; central nervous system; central neural pathway /tract; clinical research; confocal scanning microscopy; human genetic material tag; human tissue; immunocytochemistry; kernicterus; laboratory rat; neurotoxicology; newborn animals; newborn human (0-6 weeks); polymerase chain reaction; postmortem; protein localization; protein quantitation /detection; protein structure function; proteomics; retinoids; vitamin therapy

DESCRIPTION (provided by applicant): P-glycoprotein (Pgp), an ATP-binding cassette efflux pump expressed on endothelial cells and astrocytes of the bloodbrain barrier (BBB), may serve an important role in limiting the passage of bilirubin into the central nervous system (CNS). Studies performed during years 01-04 have demonstrated: i) Pgp mediated bilirubin transport in vitro; ii) bilirubin stimulated Pgp ATPase activity; iii) limited Pgp expression in the immature murine and human CNS; and iv) a marked early postnatal increase in BBB Pgp. These novel observations raise the distinct possibility that human BBB Pgp attenuates CNS bilirubin content, a phenomenon we have shown in Pgp sufficient as compared with Pgp deficient null mutant transgenic mice (167), thereby diminishing the risk for kernicterus. Preliminary studies in our lab show that Pgp i) confers cellular resistance against bilirubin-induced apoptosis, and ii) is expressed in selected cells of the CNS parenchyma in addition to those of the BBB in developing human neonates. Taken together, these findings suggest that Pgp may confer protection against bilirubin induced cell injury both at the BBB and within the CNS parenchyma. In the current proposal, we will extend our previous observations and recent preliminary findings by testing three hypotheses: 1) Pgp acts to inhibit cellular apoptosis induced by bilirubin in vitro and in vivo; 2) Pgp is expressed in cells of the CNS parenchyma as well as cells of the BBB in humans; and 3) CNS Pgp expression is upregulated by postnatal hyperbilirubinemia and antenatal maternal vitamin A treatment in Gunn rat pups, and that combined antenatal vitamin A treatment with postnatal hyperbilirubinemia will be protective against bilirubin-induced apoptosis in vivo. We will use cell lines (Caco-2, neuroblastoma, bovine brain capillary endothelial and LLC-PK1 cells tranfected with the gene for human or murine Pgp) and the Gunn rat model of neonatal jaundice to characterize the role of Pgp in protecting against bilirubin-induced apoptosis, the apoptotic pathways involved, and the contribution of apoptosis to cell death. The prevalence of apoptosis in kernicterus in human neonates, as well as the ontogeny and regional localization of human CNS Pgp expression will be assessed in archival postmortem tissue. The information obtained will provide novel insights regarding the role CNS Pgp plays in conferring resistance against kernicterus and serve as an impetus towards developing modalities that enhance CNS Pgp expression in neonates thereby affording newborns additional protection against kernicterus - a chronically disabling neurologic condition that remains of marked clinical importance both in the United States and abroad.

描述（由申请人提供）：P-糖蛋白（Pgp）是一种在血脑屏障（BBB）的内皮细胞和星形胶质细胞上表达的 ATP 结合盒外排泵，可能在限制胆红素进入中枢神经方面发挥重要作用系统（中枢神经系统）。 01-04 年进行的研究表明： i) Pgp 介导的体外胆红素转运； ii) 胆红素刺激 Pgp ATP 酶活性； iii) 在未成熟的小鼠和人类中枢神经系统中有限的 Pgp 表达； iv) 产后早期 BBB Pgp 显着增加。这些新的观察结果提出了人类 BBB Pgp 减弱 CNS 胆红素含量的明显可能性，与 Pgp 缺陷无效突变转基因小鼠相比，我们在 Pgp 中证明了这一现象（167），从而降低了核黄疸的风险。我们实验室的初步研究表明，Pgp i) 赋予细胞对胆红素诱导的细胞凋亡的抵抗力，并且 ii) 在发育中的人类新生儿中，除了 BBB 细胞外，还在 CNS 实质的选定细胞中表达。总而言之，这些发现表明 Pgp 可以在 BBB 和 CNS 实质内提供针对胆红素诱导的细胞损伤的保护。在当前的提案中，我们将通过测试三个假设来扩展我们之前的观察和最近的初步发现：1）Pgp 在体外和体内抑制胆红素诱导的细胞凋亡； 2) Pgp在人类中枢神经系统实质细胞和血脑屏障细胞中表达； 3）Gu​​nn大鼠幼仔中，产后高胆红素血症和产前母体维生素A治疗可上调中枢神经系统Pgp表达，产前维生素A治疗与产后高胆红素血症相结合可防止体内胆红素诱导的细胞凋亡。我们将使用细胞系（Caco-2、神经母细胞瘤、牛脑毛细血管内皮细胞和转染人或鼠 Pgp 基因的 LLC-PK1 细胞）和新生黄疸 Gunn 大鼠模型来表征 Pgp 在预防胆红素方面的作用。诱导细胞凋亡、涉及的细胞凋亡途径以及细胞凋亡对细胞死亡的贡献。将在档案死后组织中评估人类新生儿核黄疸中细胞凋亡的发生率以及人类 CNS Pgp 表达的个体发育和区域定位。所获得的信息将提供关于 CNS Pgp 在赋予对核黄疸抵抗力中所发挥的作用的新见解，并推动开发增强新生儿中 CNS Pgp 表达的方式，从而为新生儿提供额外的保护以预防核黄疸 - 一种慢性致残的神经系统疾病，仍然具有显着的影响。在美国和国外都具有临床重要性。