Mechanisms of extrahepatic biliary proliferation and regeneration

项目摘要/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。胆管病治疗方法的发展需要更好的 细胞和分子调控EHBD增殖的细胞分子机制 对伤害的反应。调控胆管细胞增殖的分子途径目前还不是很好。 明白了。本申请旨在回答有关EHBD再生机制的一个基本问题 并探索调控胆管上皮细胞增殖潜能的策略。该计划的基本原理 拟议的研究是定义胆管细胞反应的细胞和分子相互作用 损伤可以为胆管疾病和肝胆再生医学提供新的治疗策略。我们的 初步数据表明，Hedgehog(HH)和WNT信号在EHBD动态平衡中起重要作用 和损伤后的增殖。这一建议的主要假设是HH和WNT信号 调节上皮细胞和基质细胞之间的串扰，促进损伤后EHBD的增殖。在……里面 在这项建议中，我们将使用遗传小鼠模型和体内药物治疗。胆管结扎会 作为一种损伤模型。人和小鼠类有机物(BDO)与原代间充质共培养 细胞将被用来在体外直接研究上皮-基质串扰和通路相互作用，并增强 此项目的翻译部分。目标1将专注于HH信号并确定印度HH配体 从胆管细胞信号到GLI1-HH反应成纤维细胞间接调节胆管细胞 扩散。目标2将专注于WNT信号，并测试来自表达GLI1的细胞的WNT是否对 胆管细胞增殖。目标3将重点放在细胞-细胞和途径相互作用上，并确定IHH是否来自 胆管细胞直接调节成纤维细胞产生WNT，从而诱导胆管细胞增殖。在……下面 目标1和2我们将使用转基因报告和功能丧失的小鼠模型，药物抑制剂 HH和WNT信号、原位杂交和免疫组织化学技术确定体内 效果。在目标3下，我们采取了一种简化的方法，使用有机化合物共培养模型。它还将支持 发展强有力的新工具(转基因小鼠和器官模型)和技能(原位杂交 和流式细胞术)，以支持专注于基本信号通路的独立研究计划 调节EHBD。这一新的K08应用程序也将推动PI向独立的 NIH资助的研究人员，并支持她了解EHBD基本机制的长期目标 生物学最终改善胆管病患者的预后。

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.