Injury, progression, and fibrosis of the extrahepatic bile duct

项目摘要 胆道闭锁（BA）是一种胆管，特别是肝外胆管（EHBDs）的纤维闭塞性疾病， 折磨着世界各地的新生儿BA是儿科肝移植最常见的适应症 50%的患者在2岁时需要移植，其余大多数在成年前。的 病因和疾病的早期过程是未知的;然而，重要的最近的数据表明，BA结果 产前环境损伤（母亲除外），随后是原始损伤的进展 出生后 该提案旨在回答BA中的三个关键问题：1）为什么毒性仅限于新生儿？2)什么 决定胆管细胞损伤的修复和进展？以及3）纤维化是如何以及为什么发生在 EHBD？我们的基本假设是，这些问题的答案是在独特的功能， 新生儿胆道系统：BA是在发育过程中发生损伤的结果， 未成熟胆管的解剖特征使其易于损伤并促进 损伤和纤维化反应。我们的初步工作确定了新生儿胆管的关键特征， 潜在地增加他们对伤害的敏感性。这些包括缺乏保护性的顶端糖萼， 胆管细胞和未成熟的胆管细胞-细胞连接。我们还发现， 新生儿EHBD含有大量纤维化细胞，这些细胞已“做好准备”对损伤做出反应，并且 新生儿粘膜下层的解剖结构可能会传播损伤。我们开发了两种独特的工具， 研究导管未成熟在损伤易感性和反应中的作用。首先，我们开发了一种小鼠模型， 产前EHBD损伤。我们鉴定并合成了一种以前未知的胆固醇毒素， 胆汁酸，其在妊娠母亲处理后引起胎儿和新生小鼠中的EHBD损伤。损害 会因为胆汁酸的人性化而恶化这种模式将使我们能够使用转基因和 特别处理的小鼠，以研究新生儿导管易感因素在损伤中的重要性。二是 开发了一种微流体胆管芯片设备，使我们能够培养新生儿，成人，和遗传- 修饰的胆管细胞融合，不可渗透的单层，应用各种治疗选择性地对 顶面或底面，并确定其对关键胆管细胞功能的影响，包括 我们的三个具体目标使用这些和我们开发的其他工具来研究：1）新生儿 对损伤的易感性，包括糖萼的作用; 2）损伤进展的决定因素，包括 胆汁酸的作用;和3）EHBD的纤维化细胞的身份和粘膜下层的作用， 建筑在伤害的传播中。这项工作有可能大大改变我们对 BA并导致新的治疗选择。

PROJECT SUMMARY Biliary atresia (BA) is a fibro-obliterative disease of the bile ducts, especially the extrahepatic ducts (EHBDs), that afflicts neonates around the world. BA is the most common indication for liver transplant in the pediatric population, with 50% of patients requiring transplant by age 2 and most of the rest before adulthood. The etiology and early course of the disease are unknown; however, important recent data suggest that BA results from a prenatal environmental insult (sparing the mother) that is followed by progression of the original injury after birth. This proposal seeks to answer three key questions in BA: 1) Why is toxicity specific to neonates? 2) What determines repair versus progression of cholangiocyte injury? and 3) How and why does fibrosis occur in the EHBD? Our underlying hypothesis is that the answers to these questions are found in the unique features of the neonatal biliary system: that BA results from an injury that occurs in the context of a developmentally immature bile duct with anatomic features that make it susceptible to injury and promote progression of damage and a fibrotic response.Our preliminary work identified key features of the neonatal bile ducts that potentially increase their susceptibility to injury. These include lack of a protective apical glycocalyx on cholangiocytes and immature cholangiocyte cell-cell junctions. We also showed that the submucosa of the neonatal EHBD contains a large population of fibrogenic cells that are “primed' to respond to insults and that the anatomical structure of the neonatal submucosa may propagate injury.We developed two unique tools to study the role of duct immaturity in susceptibility and response to injury. First, we developed a mouse model of prenatal EHBD damage. We identified and synthesized a previously unknown isoflavonoid biliary toxin, biliatresone, that causes EHBD injury in fetal and neonatal mice after treatment of pregnant mothers. Damage is worsened by humanizing the bile acid profile. This model will enable us to use genetically-modified and specially-treated mice to investigate the importance of neonatal duct susceptibility factors in injury. Second, we developed a microfluidic bile duct-on-a-chip device that allows us to culture neonatal, adult, and genetically- modified cholangiocytes in a confluent, impermeable monolayer, to apply various treatments selectively to the apical or basal surface, and to determine their impact on key cholangiocyte functions, including the permeability barrier.Our three specific aims use these and other tools we have developed to study: 1) neonatal susceptibility to injury, including the role of the glycocalyx; 2) the determinants of injury progression, including the role of bile acids; and 3) the identity of the fibrogenic cells of the EHBD and the role of submucosal architecture in the spread of injury. This work has the potential to both significantly shift our understanding of BA and lead to new therapeutic options.