Neonatal Bilirubin Neurotoxicity and P-Glycoprotein

新生儿胆红素神经毒性和 P-糖蛋白

• 批准号: 6778635
• 负责人: JON F WATCHKO
• 金额: $ 28.12万
• 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/6778635
• 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），一种ATP结合盒式外排泵，该磁带在血脑屏障的内皮细胞和星形胶质细胞（BBB）上表达，可能在限制胆红素传递到中枢神经系统（CNS）方面起着重要作用。 01-04年期间进行的研究表明：i）PGP在体外介导的胆红素转运； ii）胆红素刺激PGP ATPase活性； iii）有限的PGP表达在未成熟的鼠和人类中枢神经系统中； iv）BBB PGP的产后早期增加。这些新颖的观察结果提出了一个明显的可能性，即人类BBB PGP减弱了CNS胆红素含量，这是我们在PGP中表现出的现象，与PGP缺乏无效的转基因小鼠相比（167），从而降低了对kernicterus的风险。在我们的实验室中，初步研究表明，PGP i）赋予对胆红素诱导的细胞凋亡的细胞耐药性，ii）在CNS实质的选定细胞中表达了除了发育中的人类新生儿的BBB外，还表达了对胆红素的抗性。综上所述，这些发现表明，PGP可以赋予BBB和CNS实质内胆红素诱导细胞损伤的保护。在当前的提案中，我们将通过检验三个假设来扩展以前的观察结果和最近的初步发现：1）PGP作用可抑制胆红素在体外和体内诱导的细胞凋亡； 2）PGP在CNS实质的细胞以及人类的BBB细胞中表达； 3）CNS PGP表达在Gunn大鼠幼崽中的产后多胆红素血症和产前母体维生素A治疗中上调，并且在产后高胆红素血症治疗的结合使用胆红素诱导的Vivo中的阿细胞凋亡中，这种结合使用产后高胆红素血症。我们将使用细胞系（CACO-2，神经母细胞瘤，牛脑毛细血管内皮和LLC-PK1细胞，用人或鼠PGP的基因转染了基因）以及新生儿黄疸的Gunn大鼠大鼠模型，以表征PGP在保护胆红素诱导的APOPTOSIS中的PGP作用，并涉及Apopttosis，并涉及Apopttose的促进。人类新生儿核中核细胞凋亡的患病率以及人CNS PGP表达的个体发育和区域定位将在档案后尸体组织中评估。获得的信息将提供有关中枢神经系统PGP在抗拒内核的抵抗中所扮演的作用的新见解，并成为发展模态的推动力，从而增强了新生儿中CNS PGP的表达，从而为新生儿提供了额外的保护，以防止内核 - 一种长期抑制了神经系统的持续性持续的临床和临时性的神经系统，并在联合国中均具有很大的重要性。