Elucidating the role of β-catenin signaling in liver progenitor cell-mediated liver regeneration

阐明β-连环蛋白信号在肝祖细胞介导的肝再生中的作用

• 批准号: 9395437
• 负责人: Jacquelyn Olivia Russell
• 金额: $ 4.4万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-03 至 2019-07-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9395437
• 关键词: 2-Acetylaminofluorene; Acute; Address; Albumins; American; Animals; Appearance; Biliary; Binding; Cause of Death; Cell Compartmentation; Cell Differentiation process; Cell Line; Cells; Cessation of life; Choline; Cirrhosis; Defect; Dependovirus; Development; Diet; Disease Progression; EGF gene; Enterobacteria phage P1 Cre recombinase; Epithelial Cells; Ethionine; FOXL1 gene; Genetic Models; Globulins; Hepatic; Hepatocyte; Human; Impairment; In Vitro; Injectable; Injury; Knock-out; Label; Ligands; Liver; Liver Failure; Liver Regeneration; Liver diseases; Mediating; Methods; Modeling; Mus; Natural regeneration; Organ Donor; Partial Hepatectomy; Patients; Play; Primary carcinoma of the liver cells; Protocols documentation; Rattus; Recovery; Rodent; Role; Serotyping; Severity of illness; Signal Pathway; Signal Transduction; Small Interfering RNA; Stem cells; TP53 gene; Testing; Thyroid Gland; United States; WT2 gene; Work; base; beta catenin; cholangiocyte; chronic liver disease; high risk; in vivo; knock-down; liver cell proliferation; liver injury; liver transplantation; mortality; mouse model; novel; novel therapeutics; prevent; repaired; response; senescence; targeted treatment

Project Summary/Abstract Chronic liver disease (CLD) and cirrhosis is the 12th leading cause of death in the United States. Currently, the only treatment for patients with end-stage liver disease is a liver transplant. However, the scarcity of donor organs makes this option untenable for many patients. Therefore there is a great need to develop new therapies to prevent or reverse patient progression to end-stage liver disease. Progression is thought to be due in part to failed hepatic regeneration. Under most circumstances, after liver injury hepatic repair is mediated by proliferation of hepatocytes. However, when hepatocyte proliferation is impaired, liver progenitor cells (LPCs) arise from the biliary epithelial cell (BEC) compartment, expand, and differentiate into hepatocytes. LPC expansion is observed in human CLD patients, and thus LPCs represent a promising target for therapies aimed at promoting liver regeneration (LR) in patients. However the role of LPCs in LR remains controversial. A common model to study LPCs in rodents is the choline deficient, ethionine-supplemented (CDE) diet, which induces liver injury and promotes expansion of LPCs. However, the CDE diet does not block hepatocyte proliferation, so LR is hepatocyte-driven in the CDE diet model. The β-catenin signaling pathway plays an important role in LR by promoting hepatocyte proliferation. Thus we hypothesize lack of β-catenin in hepatocytes would impair hepatocyte proliferation after CDE diet-induced liver injury and promote LPC- mediated LR. In Aim 1, we will test this hypothesis using two models of genetic fate tracing in mice. In the first model, we will perform negative lineage tracing by injecting mice with adeno-associated virus serotype 8 (AAV8) carrying Cre recombinase to simultaneously delete β-catenin in hepatocytes and label hepatocytes with EYFP. In the second method we will perform positive lineage tracing, utilizing Foxl1-Cre mice to label LPCs with EYFP and injecting these mice with β-catenin small interfering RNA conjugated to a hepatocyte- targeting ligand to knockdown β-catenin expression specifically in hepatocytes. We will place both mouse models on the CDE diet to determine if LPCs give rise to hepatocytes to mediate LR. The mechanisms of LPC differentiation to hepatocytes are also not understood. Due to the important role of β- catenin in hepatocyte maturation in development, we hypothesize β-catenin is important for LPC-to-hepatocyte differentiation. In Aim 2, we will test this hypothesis in vivo through placing mice with lack of β-catenin in both hepatocytes and BECs (the origin of LPCs) on the CDE diet, where we would expect to observe a defect in LR. We will test this hypothesis in vitro utilizing the small cholangiocyte cell line (SMCC), an immortalized BEC line which expresses LPC-marker Foxl1. We will treat SMCCs with HGF, EGF, and other factors to induce differentiation to hepatocyte-like cells, and we predict β-catenin-inhibited SMCCs will fail to differentiate. In summary, our work will thoroughly describe the role of β-catenin in LPC-mediated LR, potentially identifying a new mechanism which could be targeted to promote LR in human CLD patients.

项目总结/摘要 慢性肝病（CLD）和肝硬化是美国第12大死亡原因。目前 对终末期肝病患者的唯一治疗是肝移植。然而，捐赠者的稀缺性 器官使得这种选择对许多患者来说站不住脚。因此，非常需要开发新的 预防或逆转患者进展为终末期肝病的治疗。进展被认为是由于 部分原因是肝再生失败。在大多数情况下，肝损伤后的肝修复是由 肝细胞增殖。然而，当肝细胞增殖受损时， 从胆管上皮细胞（BEC）隔室产生、扩增并分化成肝细胞。LPC 在人类CLD患者中观察到扩增，因此LPC代表了有希望的治疗靶点 旨在促进患者的肝再生（LR）。然而，LPC在LR中的作用仍然存在争议。 研究啮齿类动物中LPC的常见模型是胆碱缺乏，乙硫氨酸补充（CDE）饮食， 诱导肝损伤并促进LPC扩增。然而，CDE饮食不会阻断肝细胞 因此，在CDE饮食模型中，LR是肝细胞驱动的。β-catenin信号通路起着 通过促进肝细胞增殖在LR中起重要作用。因此，我们假设， 肝细胞会损害CDE饮食诱导的肝损伤后的肝细胞增殖，并促进LPC- 介导的LR。在目标1中，我们将使用两种小鼠遗传命运追踪模型来验证这一假设。上 模型，我们将通过向小鼠注射腺相关病毒血清型8进行阴性谱系追踪 携带Cre重组酶的AAV 8同时缺失肝细胞中的β-连环蛋白和标记肝细胞 关于EYFP在第二种方法中，我们将进行阳性谱系追踪，利用Foxl 1-Cre小鼠标记 LPCs与EYFP，并向这些小鼠注射与肝细胞结合的β-连环蛋白小干扰RNA， 靶向配体特异性敲低肝细胞中β-连环蛋白表达。我们将把两只老鼠 CDE饮食模型，以确定LPC是否产生肝细胞介导LR。 LPC分化为肝细胞的机制也不清楚。由于β- 连环蛋白在肝细胞发育成熟过程中起重要作用，我们推测β-连环蛋白对LPC向肝细胞的转化起重要作用。 分化在目标2中，我们将通过将缺乏β-catenin的小鼠置于两个 肝细胞和BEC（LPC的来源）的CDE饮食中，我们预计会观察到LR的缺陷。 我们将利用小胆管细胞系（SMCC），一种永生化的BEC系，在体外验证这一假设 其表达LPC标记物Foxl 1。我们将用HGF、EGF和其他因子治疗SMCC， 我们预测β-catenin抑制SMCC不能分化为肝细胞样细胞。 总之，我们的工作将彻底描述β-catenin在LPC介导的LR中的作用， 一种新的机制，可以有针对性地促进人类CLD患者的LR。