Neonatal Bilirubin Neurotoxicity and P-Glycoprotein

描述（由申请人提供）：p -糖蛋白（Pgp）是一种在血脑屏障（BBB）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系统（CNS）方面发挥重要作用。在01-04年间进行的研究已经证明：i) Pgp介导的胆红素体外转运；ii)胆红素刺激Pgp atp酶活性；iii) Pgp在未成熟小鼠和人中枢神经系统中的表达有限；iv)产后早期血脑屏障Pgp显著升高。这些新的观察结果提出了人类血脑屏障Pgp降低中枢神经系统胆红素含量的明显可能性，我们已经在Pgp中证明了这一现象，与Pgp缺乏的零突变转基因小鼠相比（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)赋予细胞抵抗胆红素诱导的细胞凋亡，ii)在发育中的人类新生儿中，除了血脑屏障外，Pgp还在中枢神经系统实质的选定细胞中表达。综上所述，这些发现表明Pgp可能对胆红素诱导的血脑屏障和中枢神经系统实质细胞损伤具有保护作用。在目前的提案中，我们将扩展我们之前的观察和最近的初步发现，通过测试三个假设：1)Pgp在体外和体内抑制胆红素诱导的细胞凋亡；2) Pgp在人中枢神经实质细胞和血脑屏障细胞中均有表达；3)出生后高胆红素血症和产前母体维生素A处理上调Gunn大鼠幼鼠中枢神经系统Pgp的表达，产前维生素A与出生后高胆红素血症联合处理对体内胆红素诱导的细胞凋亡具有保护作用。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LLC-PK1细胞转染人或鼠Pgp基因）和Gunn大鼠新生儿黄疸模型来表征Pgp在防止胆红素诱导的凋亡中的作用，所涉及的凋亡途径，以及凋亡对细胞死亡的贡献。将在存档的死后组织中评估人类新生儿核内细胞凋亡的发生率，以及人类中枢神经系统Pgp表达的个体发生和区域定位。所获得的信息将为CNS Pgp在抗核黄疸中所起的作用提供新的见解，并推动开发增强新生儿CNS Pgp表达的模式，从而为新生儿提供额外的抗核黄疸保护。核黄疸是一种慢性致残的神经系统疾病，在美国和国外仍然具有显著的临床重要性。

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.