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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要目前，慢性肝病 (CLD) 和肝硬化是美国第 12 大死亡原因。终末期肝病患者的唯一治疗方法是肝移植，但供体稀缺。对于许多患者来说，这种选择是站不住脚的，因此非常需要开发新的器官。预防或逆转患者进展为终末期肝病的疗法被认为是由于。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损伤介导的。然而，当肝细胞增殖受损时，肝祖细胞（LPC）就会减少。从胆管上皮细胞 (BEC) 区室产生，扩增并分化为肝细胞。在人类 CLD 患者中观察到了扩增，因此 LPC 是一个有希望的治疗靶点然而，LPCs 在 LR 中的作用仍存在争议。研究啮齿类动物 LPC 的常见模型是缺乏胆碱、补充乙硫氨酸 (CDE) 饮食，诱导肝损伤并促进 LPC 扩张，但 CDE 饮食不会阻断肝细胞。增殖，因此 LR 在 CDE 饮食模型中是肝细胞驱动的，β-连环蛋白信号通路发挥着重要作用。通过促进肝细胞增殖在 LR 中发挥重要作用，因此我们一直在寻找 β-catenin 的缺乏。 CDE饮食诱导的肝损伤后，肝细胞会损害肝细胞增殖并促进LPC- 在目标 1 中，我们将使用两种小鼠遗传命运追踪模型来检验这一假设。模型中，我们将通过给小鼠注射腺相关病毒血清型 8 来进行负谱系追踪 (AAV8)携带Cre重组酶，同时删除肝细胞中的β-catenin并标记肝细胞在第二种方法中，我们将利用 Foxl1-Cre 小鼠进行阳性谱系追踪来进行标记。具有 EYFP 的 LPC 并向这些小鼠注射与肝细胞缀合的 β-连环蛋白小干扰 RNA 靶向配体以特异性地敲低肝细胞中的β-连环蛋白表达，我们将放置两只小鼠。 CDE 饮食模型，以确定 LPC 是否产生肝细胞来介导 LR。由于β-的重要作用，LPC分化为肝细胞的机制也不清楚。连环蛋白在肝细胞成熟发育过程中，我们认为β-连环蛋白对于LPC到肝细胞很重要在目标 2 中，我们将通过将缺乏 β-连环蛋白的小鼠置于体内来测试这一假设。 CDE 饮食中的肝细胞和 BEC（LPC 的起源），我们预计会观察到 LR 缺陷。我们将利用小胆管细胞系 (SMCC)（一种永生化 BEC 系）在体外测试这一假设 SMCC表达LPC标记Foxl1，我们将用HGF、EGF和其他因子处理SMCC以诱导。分化为肝细胞样细胞，我们预测β-连环蛋白抑制的 SMCC 将无法分化。总之，我们的工作将彻底描述 β-连环蛋白在 LPC 介导的 LR 中的作用，潜在地识别一种可以有针对性地促进人类 CLD 患者 LR 的新机制。