Mechanisms of extrahepatic biliary proliferation and regeneration

项目总结/摘要 胆管病是一种无法治愈的进行性肝外胆管（EHBD）疾病，其特征是损伤- 诱导胆管细胞过度增殖。胆管疾病治疗的发展需要更好的 了解EHBD在体内平衡过程中增殖的细胞和分子机制， 对伤害的反应。调节胆管细胞增殖的分子途径目前还不清楚。 明白本申请旨在回答EHBD再生机制的一个基本问题 并探索调节胆管上皮增殖潜力的策略。的理由 提出的研究是，定义细胞和分子相互作用的基础胆管细胞的反应， 损伤可以为胆管疾病和肝胆再生医学提供新的治疗策略。我们 初步数据表明，Hedgehog（HH）和WNT信号在EHBD稳态中起重要作用 和损伤后的增殖。该提议的首要假设是HH和WNT信号传导 调节上皮细胞和基质细胞之间的串扰，促进损伤后EHBD增殖。在 本提案中，我们将使用遗传小鼠模型和体内药物治疗。胆管结扎会 用作创伤模型。人和小鼠EHBD类器官（BDO）与原代间充质细胞共培养 细胞将被用于直接研究上皮-基质串扰和体外途径相互作用，并增强 这个项目的翻译部分。Aim 1将专注于HH信号传导，并确定印度HH配体是否 从胆管细胞向GLI 1 + HH反应性成纤维细胞发出信号，以间接调节胆管细胞 增殖目标2将集中于WNT信号传导，并测试来自GLI 1表达细胞的WNT是否对 胆管细胞增殖目标3将侧重于细胞-细胞和途径相互作用，并确定IHH是否来自 胆管细胞直接调节成纤维细胞产生WNT以诱导胆管细胞增殖。下 目的1和2，我们将使用转基因报告基因和功能丧失小鼠模型， HH和WNT信号传导、原位杂交和免疫组织化学技术，以确定体内 方面的影响.在目标3下，我们通过使用类器官共培养模型采取简化方法。它还将支持 开发强大的新工具（转基因小鼠和类器官模型）和技能（原位杂交 和流式细胞术），以支持一个专注于基本信号通路的独立研究计划 调节EHBD。这一新的K 08应用程序也将促进PI发展成为一个独立的 NIH资助的研究人员，并支持她的长期目标，以了解EHBD的基本机制 生物学，以最终改善胆管疾病患者的预后。

PROJECT SUMMARY/ABSTRACT Cholangiopathies are incurable, progressive extrahepatic bile duct (EHBD) disorders characterized by injury- induced cholangiocyte hyperproliferation. Development of therapies for cholangiopathies requires better understanding of cellular and molecular mechanisms regulating EHBD proliferation during homeostasis and in response to injury. Molecular pathways that regulate cholangiocyte proliferation are currently not well understood. This application aims to answer a fundamental question about mechanisms of EHBD regeneration and explore strategies to modulate the proliferative potential of the biliary epithelium. The rationale for the proposed research is that defining cellular and molecular interactions underlying cholangiocyte responses to injury can offer novel therapeutic strategies for cholangiopathies and hepatobiliary regenerative medicine. Our preliminary data suggest that Hedgehog (HH) and WNT signaling play important roles in EHBD homeostasis and proliferation after injury. The overarching hypothesis for this proposal is that HH and WNT signaling regulate crosstalk between epithelial and stromal cells to promote EHBD proliferation after injury. In this proposal, we will use genetic mouse models and pharmacological treatments in vivo. Bile duct ligation will be used as an injury model. Human and mouse EHBD organoid (BDO) co-culture with primary mesenchymal cells will be used to directly study epithelial-stromal crosstalk and pathway interaction in vitro and enhance the translational component of this project. Aim 1 will focus on HH signaling and determine if the Indian HH ligand from cholangiocytes signals to GLI1+ HH-responsive fibroblasts to indirectly regulate cholangiocyte proliferation. Aim 2 will focus on WNT signaling and test if WNT from GLI1-expresing cells is critical for cholangiocyte proliferation. Aim 3 will focus on cell-cell and pathway interactions and determine if IHH from cholangiocytes directly regulates WNT production by fibroblasts to induce cholangiocyte proliferation. Under Aims 1 and 2 we will use transgenic reporter and loss-of-function mouse models, pharmacological inhibitors of HH and WNT signaling, in situ hybridization, and immunohistochemistry techniques to define the in vivo effects. Under Aim 3 we take a reductionist approach by using organoid co-culture models. It will also support the development of robust new tools (transgenic mice and organoid models) and skills (in situ hybridization and flow cytometry) to support an independent research program focused on fundamental signaling pathways regulating EHBD. This new K08 application will also promote the development of the PI into an independent NIH-funded investigator and support her long-term goal to understand fundamental mechanisms of EHBD biology to ultimately improve outcomes in patients with cholangiopathies.