Injury, progression, and fibrosis of the extrahepatic bile duct

项目总结 胆道闭锁(BA)是一种胆管，尤其是肝外胆管的纤维闭塞性疾病。 这困扰着世界各地的新生儿。BA是小儿肝移植最常见的适应症 人口，50%的患者在2岁之前需要移植，其余大部分在成年前。这个 疾病的病因和早期病程尚不清楚；然而，最近重要的数据表明，BA的结果 来自产前环境的侮辱(不伤害母亲)，随后是原始损伤的进展 出生后。 该提案试图回答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还是进展？3)肝纤维化是如何发生的，为什么发生在 EHBD？我们的基本假设是，这些问题的答案存在于 新生儿胆道系统：BA是在发育过程中发生的损伤引起的 具有解剖特征的未成熟胆管，使其易受损伤并促进胆管病变的进展 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 潜在地增加了他们对伤害的敏感性。这些问题包括缺乏保护性的顶端糖基化 胆管细胞和未成熟的胆管细胞-细胞连接。我们还发现，粘膜下层的 新生儿EHBD包含大量的纤维化细胞，这些细胞对侮辱做出反应，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来 研究导管不成熟在损伤易感性和反应中的作用。首先，我们开发了一个小鼠模型 胎儿期高血压病的损害。我们鉴定并合成了一种未知的异黄酮类胆汁毒素， 胆红素，在怀孕母亲治疗后，会对胎儿和新生小鼠造成EHBD损伤。毁伤 因为胆汁酸的人性化而变得更糟。这种模式将使我们能够使用转基因和 用特殊处理的小鼠研究新生小鼠胆管损伤易感因素的重要性。第二，我们 开发了一种芯片上的微流控胆管设备，允许我们培养新生儿、成人和基因- 在融合的、不渗透的单层中修饰胆管细胞，选择性地将各种治疗应用于 并确定它们对关键的胆管细胞功能的影响，包括 渗透性屏障。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，包括糖基化酶的作用；2)损伤进展的决定因素，包括 胆汁酸的作用；以及3)EHBD纤维化细胞的特性和粘膜下的作用 建筑中的伤痕蔓延。这项工作有可能极大地改变我们对 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.