Identification, Quantification, and Functional Characterization of Transporters in Human Placenta, Developing Gut and Fetal Brain

摘要 胎盘，通常被称为胎儿的“肠”，是控制 营养素(包括维生素)和外源物质(即膳食补充剂和FDA批准的药物)之间 母亲和她的胎儿。胎儿也可以通过消化道摄取营养物质和外来物质，新生儿也是如此。 和婴儿出生后通过母乳喂养。胎儿、新生儿和婴儿的血脑屏障(BBB)具有 在保护发育中的大脑免受外来物质的伤害和向大脑提供营养方面起着至关重要的作用。因此， 胎盘、发育中的血脑屏障和肠道是负责营养和异种生物分布和 吸收对早期人类发育和异种生物毒性的影响。运输者可以扮演一个必不可少的角色 在营养物质和外来物质的吸收、全身暴露和组织分布中的作用 胎儿、新生儿和婴儿跨越胎盘、肠道和血脑屏障。身份识别和 这些组织屏障中转运蛋白的定量对于了解和预测胎儿或 新生儿/婴儿对营养物质和外来物质的摄取和接触，从而影响早期发育，如 以及在这些弱势群体中安全有效地使用药物/补充剂。而当 几种ABC转运蛋白在人足月胎盘中的表达及功能 乳腺癌耐药蛋白(BCRP)，已经被很好地描绘出来，这样的数据对于 转运蛋白在妊娠早期胎盘、发育中的肠道和妊娠期间和出生后的血脑屏障中。在……里面 在这项申请中，我们建议在华盛顿大学建立转运蛋白阐明中心(TEC) 这解决了RFA-HD-23-003中阐明的目标。利用定量的全球和靶向蛋白质组学， 我们将系统地鉴定和量化人类胎盘转运蛋白的个体发育(从早期开始 妊娠至足月)、发育中的肠道以及发育中的血脑屏障(从妊娠早期、中期及以后 出生)。然后，通过体外(转运蛋白转染细胞、免疫组织化学、免疫定位)和EX 体内转运(例如，胎盘灌流、肠道器官和IPSC来源的人胎儿血脑屏障模型) 研究中，我们将确定新的底物、细胞定位和高度丰富的转运活性 这些组织中的转运体。总而言之，这些研究将解决我们在 对转运蛋白在体内控制基本生理功能和异种生物处置的理解 发育中的胎儿和新生儿/婴儿。因此，建议的研究将加强我们预测 外源物质的毒性或有效性以及营养物质(或缺乏营养物质)对这些脆弱人群的生理效果 人口。

SUMMARY The placenta, often referred to as the “intestine” of the fetus, is an essential organ that controls the exchange of nutrients (including vitamins) and xenobiotics (i.e., dietary supplements and FDA approved drugs) between the mother and her fetus. The fetus can also ingest nutrients and xenobiotics via the digestive tract, so do neonates and infants through breastfeeding after birth. The fetal, neonate, and infant blood-brain barrier (BBB) serves a critical role in protecting the developing brain from xenobiotics and supplying nutrients to the brain. Thus, the placenta, the developing BBB and gut are key organs responsible for nutrient and xenobiotic distribution and absorption impacting early human development and xenobiotic toxicity. Transporters can play an essential role in the absorption, systemic exposure, and tissue distribution of nutrients and xenobiotics in the fetus, neonates, and infants across the placental, intestinal, and blood-brain barriers. Identification and quantification of transporters in these tissue barriers is important for understanding and predicting fetal or neonate/infant uptake of, and exposure to, nutrients and xenobiotics, and hence impacting early development as well as the safe and efficacious use of medications/supplements in these vulnerable populations. While the expression and function of a few ABC transporters in human term placenta, such as P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), have been well-delineated, such data are sorely missing for transporters in early gestation placenta, and in the developing gut and BBB during pregnancy and after birth. In this application, we propose to establish a Transporter Elucidation Center (TEC) at the University of Washington that addresses the goals articulated in RFA-HD-23-003. Using quantitative global and targeted proteomics, we will systematically identify and quantify the ontogeny of transporters in the human placenta (from early gestation to term), the developing gut as well as the developing BBB (from early and mid-gestation and after birth). Then, through in vitro (transporter-transfected cells, immunohistochemistry, immunolocalization) and ex vivo (e.g., placental perfusion, intestinal organoids, and iPSC-derived human fetal BBB models) transport studies, we will determine novel substrates, cellular localization, and transport activity of highly abundant transporters in these tissues. Combined, these studies will address a critical knowledge gap in our understanding of transporters that control essential physiological functions and xenobiotic disposition in the developing fetus and neonate/infant. Consequently, the proposed studies will enhance our ability to predict the toxicity or efficacy of xenobiotics and physiological efficacy of nutrients (or lack thereof) in these vulnerable populations.