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.

项目摘要/摘要 慢性肝病（CLD）和肝硬化是美国第十二个主要死亡原因。目前， 终末期肝病患者的仅治疗是肝移植。但是，捐助者的稀缺性 器官使许多患者无法实现此选项。因此，需要开发新的 预防或逆转患者发展为末期肝病的疗法。人们认为进步是到期的 部分原因是肝脏再生失败。在大多数情况下，肝损伤后肝修复介导 肝细胞的扩散。但是，当肝细胞增殖受到损害时，肝脏祖细胞（LPC） 由胆道上皮细胞（BEC）室膨胀并分化为肝细胞。 LPC 在人类CLD患者中观察到膨胀，因此LPC代表了治疗的前景靶标 旨在促进患者的肝脏再生（LR）。但是，LPC在LR中的作用仍然存在争议。 在啮齿动物中研究LPC的一个常见模型是缺乏胆碱的埃塞三宁饮食（CDE）饮食 诱导肝损伤并促进LPC的扩张。但是，CDE饮食不会阻止肝细胞 增殖，因此在CDE饮食模型中是肝细胞驱动的。 β-catenin信号通路播放 通过促进肝细胞增殖在LR中的重要作用。我们假设缺乏β-catenin 肝细胞会损害CDE饮食诱导肝损伤后肝细胞的增殖并促进LPC- 介导的LR。在AIM 1中，我们将使用小鼠中的两个遗传命运追踪模型检验这一假设。在第一个 模型，我们将通过向小鼠注射腺相关病毒血清型8来执行阴性谱系跟踪 （AAV8）携带CRE重组酶简单地删除肝细胞和标签肝细胞中的β-catenin 与EYFP。在第二种方法中，我们将使用FOXL1-CRE小鼠进行标记，我们将执行正谱系跟踪 LPC与EYFP，并向这些小鼠注射β-catenin的小干扰RNA，将RNA偶联到肝细胞 靶向配体以敲低β-catenin在肝细胞中的表达。我们将把两个鼠标放在 CDE饮食上的模型以确定LPC是否引起肝细胞介导LR。 LPC分化为肝细胞的机制也不清楚。由于β-的重要作用 链球菌在发育中的肝细胞成熟中，我们假设β-catenin对LPC至 - 肝细胞很重要 分化。在AIM 2中，我们将通过将小鼠放置在两者中缺乏β-catenin的小鼠中检验该假设 CDE饮食中的肝细胞和BEC（LPC的起源），我们期望在LR中观察到缺陷。 我们将使用小胆管细胞系（SMCC）在体外检验该假设，这是一种永生的BEC线 表达LPC-Marker Foxl1。我们将使用HGF，EGF和其他因素来治疗SMCC来诱导 与肝细胞样细胞的分化，我们预测抑制β-catenin抑制的SMCC将无法区分。 总而言之，我们的工作将彻底描述β-catenin在LPC介导的LR中的作用，有可能识别 一种可以促进人类CLD患者LR的新机制。