Gene Expression Pattern of Liver Stem/Progenitor Cells

描述（由申请人提供）：这项工作是基于这样的假设，即胎儿肝脏中含有一种未达到终末分化的肝干/祖细胞亚群，在成人静止肝脏中作为休眠/慢循环、无法识别的干细胞或部分分化的祖细胞而停止。在一定条件下，当肝实质大面积受损时，肝干/祖细胞可被激活，以未知机制增殖分化为成熟肝细胞和胆道上皮细胞，重新填充肝脏，或者发生恶性转化。由于肝干/祖细胞的生物学和病理生物学意义和适用性，我们的长期目标是：(1)从胎儿和成人肝脏中鉴定和分离这些细胞，(2)在培养中维持/繁殖肝干/祖细胞，(3)阐明导致其激活、增殖和分化的分子机制以及参与这些过程的基因。为了实现这些目标，基因分析的现代方法，抑制减法杂交（SSH）和cDNA微阵列技术，正在被利用。利用SSH技术已鉴定出5个胎儿肝上皮细胞特异性克隆，并将进一步分析。利用cDNA微阵列技术鉴定和分离肝干细胞和祖细胞的标记物。研究胚胎肝上皮细胞在体内的基因表达谱，并将获得的克隆按功能群和簇进行分类。那些与上皮细胞生长、增殖和分化有关的基因将进行功能分析。所鉴定的标记的细胞特异性及其对分离靶细胞的适用性将采用单个细胞中基因表达的原位分析技术进行测试。研究不同生长因子及ECM蛋白tenascin C（观察ED13胎肝诱导表达）对培养的胎肝上皮细胞维持、增殖和分化的生物学功能。在完成肝干细胞基因表达谱的实验后，将绘制出肝干细胞活化、增殖和分化的分子模型。肝干细胞和祖细胞特异性标记的鉴定将使这些细胞的分离，并与特定的生长因子/细胞因子和细胞外基质蛋白的知识，必要的他们的生长和分化，他们的体外繁殖。

DESCRIPTION (provided by applicant): This work is based on the hypothesis that the fetal liver contains a subset of hepatic stem/progenitor cells that have not reached terminal differentiation and arrest in the adult quiescent liver as dormant/slow cycling, unidentifiable stem cells or partially differentiated progenitor cells. Under certain conditions, when the liver parenchyma is extensively damaged, liver stem/progenitor cells can be activated by yet unknown mechanisms to proliferate and differentiate into mature hepatocytes and biliary epithelial cells and repopulate the liver or, alternatively, undergo malignant transformation. Because of the biological and pathobiological significance and applicability of liver stem/progenitor cells, our long-term objectives are: (i) identification and isolation of these cells from fetal and adult liver, (ii) maintenance/propagation of liver stem/progenitor cells in culture, and (iii) elucidation of molecular mechanisms leading to their activation, proliferation and differentiation and the genes involved in these processes. To achieve these goals, modern methods of gene analysis, suppression-subtractive hybridization (SSH) and cDNA microarray technology, are being utilized. Five fetal liver epithelial cell specific clones have been identified with the SSH technology and will be analyzed further. Markers for identification and isolation of liver stem and progenitor cells will be identified and isolated using cDNA microarrays. Gene expression profiles of differentiating fetal liver epithelial cells in vivo will be studied and the clones obtained sorted into functional groups and clusters. Those implicated in epithelial cell growth, proliferation and differentiation will be subjected to functional analysis. The cell specificity of the identified markers and their applicability for isolation of the target cells will be tested employing in situ technique of analysis of gene expression in the individual cells. The biological function of different growth factors and the ECM protein tenascin C (for which induced expression in ED13 fetal liver was observed), on maintenance, proliferation and differentiation of fetal liver epithelial cells in culture will be studied. Upon completion of the experiments on the gene expression profile of liver stem cells, a molecular model of activation, proliferation and differentiation of these cells will be drawn. The liver stem and progenitor cell specific markers identified will enable the isolation of these cells and with the knowledge of the specific growth factors/cytokines and extracellular matrix proteins necessary for their growth and differentiation, their propagation in vitro.