Gene Expression Pattern of Liver Stem/Progenitor Cells

描述(申请人提供)：这项工作是基于这样的假设，即胎儿肝脏含有一组尚未达到终末分化的肝脏干细胞/前体细胞，在成人静止肝脏中作为休眠/缓慢循环、无法识别的干细胞或部分分化的前体细胞而停止。在某些条件下，当肝实质广泛受损时，肝干/祖细胞可以通过未知的机制被激活，增殖和分化为成熟的肝细胞和胆管上皮细胞，并重新填充到肝脏，或者，经历恶性转化。由于肝干/祖细胞的生物学和病理生物学意义及其适用性，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，人们正在利用现代的基因分析方法，如抑制消减杂交(SSH)和基因芯片技术。利用SSH技术已鉴定出5个胎肝上皮细胞特异性克隆，并将对其进行进一步分析。用于识别和分离肝脏干细胞和祖细胞的标记将使用基因芯片进行识别和分离。将研究体内分化的胎肝上皮细胞的基因表达谱，并将获得的克隆分类为功能基团和簇。那些与上皮细胞生长、增殖和分化有关的基因将接受功能分析。将使用单个细胞中基因表达的原位分析技术来测试所识别的标记的细胞特异性及其对分离靶细胞的适用性。研究不同的生长因子和ECM蛋白Tenascin C(在ED13胎肝中诱导表达)对胎肝上皮细胞的维持、增殖和分化的生物学作用。在完成肝干细胞基因表达谱实验后，将绘制出这些细胞激活、增殖和分化的分子模型。确定的肝干细胞和祖细胞特异性标志物将有助于分离这些细胞，并了解它们生长和分化所需的特定生长因子/细胞因子和细胞外基质蛋白，以及它们的体外繁殖。

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.