Identifying mechanisms of liver stem cell proliferation and differentiation

鉴定肝干细胞增殖和分化的机制

• 批准号: 7449294
• 负责人: Carl Barth Rountree
• 金额: $ 14.93万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7449294
• 关键词: Albumins; Animal Model; Biological Assay; Cell Cycle; Cell Differentiation process; Cell Proliferation; Cell Therapy; Cell surface; Cells; Chronic; Development; Diet; Elements; Environment; Epidermal Growth Factor; Event; Fetal Liver; Flow Cytometry; Foundations; Future; Gene Expression; Genetic Predisposition to Disease; Goals; Growth Factor; Growth Factor Receptors; Hepatocyte; Hepatocyte Growth Factor; Human; In Vitro; Indium; Injury; Injury to Liver; Investigation; KRT19 gene; Keratin-19; Knock-out; Lead; Liver; Liver Stem Cell; Liver diseases; Modeling; Molecular; Molecular Profiling; Multiple Trauma; Mus; Natural regeneration; Nuclear; Nuclear Protein; Nuclear Proteins; Oxidative Stress; Phenotype; Phosphorylation; Polymerase Chain Reaction; Population; Population Control; Proliferating; Protein Analysis; Public Health; Publishing; Reporting; Research; Research Personnel; Role; Small Interfering RNA; Stem cells; Surface; Testing; Tissues; Transcriptional Activation; Up-Regulation; Work; Zalcitabine; adult stem cell; base; cholangiocyte; in vivo; oval cell; programs; promoter; receptor expression; response; stellate cell; stem; transcription factor

DESCRIPTION (provided by applicant): Liver stem cells, or oval cells, proliferate in response to chronic liver injury and are believed to differentiate into both hepatocytes and cholangiocytes. We have recently identified a population of CD133+ murine oval cells that demonstrates bi-lineage (i.e. hepatocyte and cholangiocyte) gene expression at the single cell level. In this proposal, we will investigate oval cells phenotypes across multiple murine injury models. Our goal is to identify reliable oval cell immuno-phenotypes, so that we can further dissect the factors that influence proliferation and differentiation. Our hypothesis is that chronic liver injury results in growth factor stimulated proliferation and activates key factors in oval cell differentiation. We will utilize three complementary models of liver injury: 1) chronic oxidative stress (MAT1a KO), 2) environmentally-induced damage (DDC 0.1% diet), and 3) a tissue specific induced regulatory knockout (Pten liver KO). To test our hypothesis, we developed the following specific aims. Aim 1: Identify the oval cell phenotype in three models of chronic liver injury. Flow cytometry isolated CD133+ oval cells will be defined by bi-lineage potential and stem cell expression profile (Albumin, aFP, Ck19 and Hnf4a). Single cell in-vitro clonal analysis will document bi-potency. We will perform in-vivo functional analysis using bulk populations. Aim 2: Define the oval cell response to growth factors stimulation. Using quantitative PCR, we will demonstrate up-regulation of growth factor receptor expression. Using phospho-protein analysis, we will identify the intra-cellular phosphorylation events in growth factor stimulated oval cells. Lastly, we will define the response to growth factor receptor blockade in-vivo within oval cell population using cell proliferation assays. Aim 3: Dissect the role of transcription factor induced differentiation within populations of proliferating oval cells. We will downregulate HNF4a using siRNA to block differentiation in-vitro. We will isolate nuclear proteins from proliferating oval cells and control populations to define transcription factor profile important to oval cell differentiation. In terms of the relevance to public health, this research will provide the cornerstone of future developments to define the potential of adult stem cells in cellular based therapies for human liver disease. This proposal identifies the three key elements to oval cell based therapy: 1) reliable identification, 2) mechanisms of proliferation, and 3) key factors in differentiation.

描述（由申请人提供）： 肝干细胞或卵圆细胞在慢性肝损伤时增殖，并被认为分化为肝细胞和胆管细胞。我们最近鉴定了一群CD 133+鼠卵圆细胞，其在单细胞水平上表现出双系（即肝细胞和胆管细胞）基因表达。在这个建议中，我们将研究卵圆细胞表型在多种小鼠损伤模型。我们的目标是确定可靠的卵圆细胞免疫表型，以便我们可以进一步剖析影响增殖和分化的因素。我们的假设是，慢性肝损伤导致生长因子刺激的增殖和激活卵圆细胞分化的关键因素。我们将利用三种互补的肝损伤模型：1）慢性氧化应激（MAT 1a KO），2）环境诱导的损伤（DDC 0.1%饮食），3）组织特异性诱导的调控敲除（Pten肝脏KO）。为了验证我们的假设，我们制定了以下具体目标。目的1：鉴定三种慢性肝损伤模型中卵圆细胞的表型。流式细胞术分离的CD 133+卵圆细胞将通过双谱系潜力和干细胞表达谱（白蛋白、aFP、Ck19和Hnf4a）来定义。单细胞体外克隆分析将记录双效价。我们将使用大量群体进行体内功能分析。目的2：确定卵圆细胞对生长因子刺激的反应。使用定量PCR，我们将证明生长因子受体表达的上调。利用磷蛋白分析，我们将确定在生长因子刺激的卵圆细胞的细胞内磷酸化事件。最后，我们将使用细胞增殖测定来确定卵圆细胞群内对生长因子受体阻断剂的体内反应。目的3：探讨转录因子在卵圆细胞增殖分化中的作用。我们将使用siRNA下调HNF4a以阻断体外分化。我们将从增殖的卵圆细胞和对照人群中分离核蛋白，以确定对卵圆细胞分化重要的转录因子谱。就与公共卫生的相关性而言，这项研究将为未来的发展提供基石，以确定成体干细胞在人类肝病细胞治疗中的潜力。该提案确定了基于卵圆细胞的治疗的三个关键要素：1）可靠的识别，2）增殖机制，以及3）分化的关键因素。