Mechanisms of extrahepatic biliary proliferation and regeneration

项目总结/摘要 该申请寻求对正在进行的NIDDK K08资助项目的行政补充。这种支持 将确保完成拟议的研究项目与新的，免费的国家的最先进的方法 并促进PI过渡到下一个职业阶段，一个独立的NIH资助的调查员。的 补充将提供所需的资金，以支持在PI的关键生活事件，分娩和 对母亲的首要责任。该研究项目的重点是胆管疾病， 是无法治愈的进行性肝外胆管（EHBD）疾病，其特征是损伤诱导的 炎症和胆管细胞过度增殖。胆管疾病治疗的发展需要 更好地理解在稳态过程中调节EHBD增殖的细胞和分子机制 以及对损伤的反应。这个补充和父应用程序的目的是回答基本问题 关于EHBD再生的机制和探索调节胆管增殖的策略。的 提出这项研究的基本原理是， 对损伤的反应可以为胆管疾病和肝胆再生的新的治疗策略提供信息。 药我们的研究结果表明，Hedgehog（HH）和WNT信号在EHBD中起重要作用 稳态和对损伤的反应。急性损伤后胆管细胞增殖增加与 胆管细胞HH配体表达的上调，其诱导CXCL 1的基质细胞表达。的 CXCL1细胞因子反过来将中性粒细胞募集到损伤组织以促进胆管增殖。此外，本发明还 EHBD损伤导致体内WNT信号上调，需要WNT通路放大器RSPO 体外EHBD类器官增殖。这一点和父提案的首要假设是，HH 和WNT信号调节上皮细胞和基质细胞之间的串扰以促进EHBD 损伤后增殖。在本研究中，我将使用野生型和基因报告小鼠来研究EHBD细胞 人口。胆管结扎将被用作损伤模型。我将检查EHBD细胞表达谱， 体内平衡和损伤后使用单细胞和批量RNA测序方法。这些研究将有助于 (1)确定GLI1 + HH-反应性细胞是否是表达Cxc11的成纤维细胞;（2）确定GLI1+细胞是否是表达Cxc11的成纤维细胞; RSPO的来源;（3）了解梗阻性损伤诱导的上皮、间质和 EHBDs中的免疫细胞群。这些实验与母体的目标1和2直接相关 并将增加K08项目成果的影响。重要的是，它们还将产生初步的 成功的R01应用程序的数据。最终，补充将使PI加快势头， 促进她转变为独立的NIH资助的研究者，并支持她的长期目标， 了解EHBD生物学的基本机制，以最终改善患者的结局， 胆管疾病

PROJECT SUMMARY/ABSTRACT This application seeks an administrative supplement for the ongoing NIDDK K08 funded project. This support will assure completion of the proposed research project with new, complimentary state-of-the-art approaches and facilitate the PI’s transition to the next career stage, an independent NIH-funded investigator. The Supplement will provide needed funds to support progress during the PI’s critical life events, childbirth and primary caregiving responsibilities for her mother. The research project is focused on cholangiopathies, which are incurable, progressive extrahepatic bile duct (EHBD) disorders characterized by injury-induced inflammation and cholangiocyte hyperproliferation. Development of therapies for cholangiopathies requires a better understanding of cellular and molecular mechanisms regulating EHBD proliferation during homeostasis and in response to injury. This supplement and the parent application aim to answer fundamental questions about the mechanisms of EHBD regeneration and explore strategies to modulate biliary proliferation. The rationale for the proposed research is that defining cellular and molecular crosstalk underlying cholangiocyte responses to injury can inform novel therapeutic strategies for cholangiopathies and hepatobiliary regenerative medicine. Our findings to date suggest that Hedgehog (HH) and WNT signaling play important roles in EHBD homeostasis and response to injury. Increased cholangiocyte proliferation after acute injury is associated with an upregulation of cholangiocyte HH ligand expression, which induces stromal cell expression of CXCL1. The CXCL1 cytokine, in turn, recruits neutrophils to the injured tissue to promote biliary proliferation. Additionally, EHBD injury results in WNT signaling upregulation in vivo, and the WNT pathway amplifier, RSPO, is required for EHBD organoid proliferation in vitro. The overarching hypothesis for this and the parent proposal is that HH and WNT signaling regulate crosstalk between epithelial and stromal cells to promote EHBD proliferation after injury. In this proposal I will use wild type and genetic reporter mice to study EHBD cell populations. Bile duct ligation will be used as an injury model. I will examine EHBD cell expression profiles at homeostasis and after injury using single cell and bulk RNA sequencing approaches. These studies will help to (1) determine if GLI1+ HH-responsive cells are fibroblasts expressing Cxcl1; (2) determine if GLI1+ cells are a source of RSPO; and (3) gain insight in obstructive injury-induced expression profiles of epithelial, stromal, and immune cell populations in EHBDs. These experiments are directly pertinent to Aims 1 and 2 of the parent proposal and will increase impact of the K08 project results. Importantly, they will also generate preliminary data for a successful R01 application. Ultimately, the Supplement will allow the PI to step up momentum and promote her transition 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.