Developmental Regulation of Drug Processing Genes

描述(申请人提供)：儿童特别容易受到药物和环境化学品的不良影响，因为他们处理外来物质的能力还不成熟。在父母R01拨款的支持下，研究人员正在积极研究小鼠药物加工基因(DPG)的发育调节。他们的长期目标是了解异种代谢和肝脏毒性方面的物种差异和相似之处，这是药物开发和人类暴露于环境化学品的风险评估中的一个巨大挑战。这项建议的目标是招募在干细胞/癌症研究、生物工程和生物信息学方面具有专业知识的新合作者，以建立一个跨学科团队和一个虚拟财团，以开发干细胞分化和成熟的新型3D肝细胞培养模型，以弥合将科学成果从动物转化为人类的巨大差距。目前干细胞向肝细胞体外分化的瓶颈是分化后的肝细胞多为低表达DPG的未成熟肝细胞，这就需要对异种代谢和肝毒性进行评估。3D培养为细胞分化和肝细胞功能提供了必要的微环境。生长激素(GH)在出生后肝脏发育中起着关键作用。氧的可获得性是干细胞分化和肝细胞代谢功能的关键。缺氧诱导因子1a(HIF-1？)和核因子-β是低氧反应的主要调节因子。肝细胞核因子4？(HNF4？)是肝细胞分化和肝功能的主要调节者。β-连环蛋白不仅在肝脏形态发生中起关键作用，而且在肝脏的基础表达和DPG的诱导中也起着关键作用。HIF-1、NF-β、HNF4和β-catenin在基因调控中存在广泛的相互作用，但它们在肝脏发育过程中如何相互作用和调控肝细胞的分化和成熟尚不清楚。核心假设是，通过影响HIF-1、NF-β、HNF4、Stat5、β-catenin和β-catenin效应器Lef-1之间的相互作用，三维微环境、氧气的可获得性和生长激素是肝细胞成熟所必需的。这一中心假设将在三个具体目标中得到检验。目的1利用芯片测序技术研究HIF-1、NF-β、HNF4、STAT5、β-catenin和Lef-1在小鼠肝细胞分化成熟过程中调控基因表达的相互作用及三维培养模型。目标2将决定HNF4之间的物理相互作用？和Lef-1/TCF4及β-连环蛋白调节剂对小鼠干细胞肝细胞分化成熟的影响。目的3将确定3D纳米纤维支架、氧分压和生长激素对小鼠干细胞分化的肝细胞成熟的影响。这一新的干细胞肝细胞分化和成熟的三维培养模型将是阐明人类和小鼠在以下方面的物种差异和相似之处的宝贵工具：1)DPG的发育调节；2)环境化学物质和治疗药物在肝脏发育过程中对肝细胞分化和成熟的影响；以及3)外源物质的代谢和肝脏毒性。

DESCRIPTION (provided by applicant): Children are particularly susceptible to adverse effects of drugs and environmental chemicals due to their immature ability to process xenobiotics. Supported by the parent R01 grant, the investigators are actively studying developmental regulation of drug processing genes (DPGs) in mice. Their long-term goal is to understand species differences and similarities in xenobiotic metabolism and hepatotoxicity, a huge challenge in drug development and risk assessment of human exposure to environmental chemicals. The objective of this proposal is to recruit new collaborators with expertise in stem-cell/cancer research, bioengineering, and bioinformatics to establish a transdisciplinary team and a virtual consortium to develop a novel 3D culture model of hepatocyte differentiation and maturation from stem cells to bridge the huge gap in translating scientific findings from animals to humans. The current bottleneck in hepatocytes differentiation from stem cells in vitro is that the differentiated hepatocytes are largely immature hepatocytes with very low expression of DPGs, which are required to evaluate xenobiotic metabolism and hepatotoxicity. 3D culture provides a microenvironment essential for cell differentiation and hepatocyte function. Growth hormone (GH) plays a key role in postnatal liver development. Oxygen availability is essential in differentiation of stem cells and metabolic function of hepatocytes. Hypoxia-inducible factor 1a (HIF-1?) and NF-?B are master regulators of hypoxic response. Hepatocyte nuclear factor 4? (HNF4?) is a master regulator of hepatocyte differentiation and liver function. ?-catenin plays a key role in not only liver morphogenesis, but also hepatic basal expression and induction of DPGs. There are extensive interactions among HIF-1?, NF-?B, HNF4?, and ?-catenin in gene regulation; however, how they interact and regulate hepatocyte differentiation and maturation during liver development is unknown. The central hypothesis is that 3D microenvironment, oxygen availability, and GH are essential in hepatocyte maturation via affecting interactions among HIF-1?, NF-?B, HNF4?, Stat5, ?-catenin, and the ?-catenin effector LEF-1. This central hypothesis will be tested in 3 specific aims. Aim 1 will use ChIP- sequencing to determine the interactions among HIF-1?, NF-?B, HNF4?, Stat5, ?-catenin, and LEF-1 in regulating gene expression during hepatocyte differentiation and maturation in mouse liver and a 3D culture model. Aim 2 will determine the physical interaction between HNF4? and LEF-1/TCF4 and effects of ?-catenin modulators on the differentiation and maturation of hepatocytes from mouse stem cells. Aim 3 will determine effects of a 3D nanofibrous scaffold, oxygen tension, and GH on the maturation of hepatocytes differentiated from mouse stem cells. This novel 3D culture model of hepatocyte differentiation and maturation from stem cells will be an invaluable tool to elucidate species differences and similarities between humans and mice in: 1) developmental regulation of DPGs; 2) effects of environmental chemicals and therapeutic drugs on hepatocyte differentiation and maturation during liver development; and 3) metabolism and hepatotoxicity of xenobiotics.