Protein Synthesis in Normal and Regenerating Liver

描述(申请人提供)：目前，全肝移植是治疗终末期慢性肝功能衰竭的主要方法。随着可用的供体肝脏的明显短缺，人们对识别干细胞或祖细胞可以恢复肝脏质量和功能有相当大的兴趣。我们已经证明，胎肝上皮细胞可以在正常肝脏中重新填充高达30%的实质肿块，产生新的肝细胞和胆管，并在细胞移植后持续增殖长达一年。这些都是干细胞的特性，我们将这些细胞称为胎肝干细胞/祖细胞(FLSPC)。我们假设，使用正常的基于肝脏的细胞移植系统，例如我们建立的系统，对于确定分离的细胞和细胞系的干细胞潜力以及确定有助于它们在肝脏中增殖和分化的基本因素是至关重要的。最近，我们发现胎肝细胞通过细胞竞争取代宿主肝细胞，这一过程最初是在果蝇中描述的。在此背景下，实验被提出：1.研究胎肝干细胞/祖细胞植入肝实质是肝脏再生的限速步骤的假说。这些研究将利用小鼠和大鼠胎肝干/祖细胞，这些细胞已经使用免疫磁珠(MAC)纯化到95%的同质性。2.研究调节细胞竞争的假设，了解细胞竞争发生的环境将导致确定移植的FlsPC进行肝脏再繁殖的最佳条件。人口再分配/竞争还会持续多久？细胞移植时宿主的年龄是否会影响再繁殖的程度？竞争总是由宿主肝脏中增加的细胞凋亡推动的吗？我们能否在移植前通过下调抗凋亡基因Survivin的表达来阻止肝脏再繁殖？3.确定哺乳动物肝脏再繁殖过程中调节细胞竞争的特定基因、信号通路和机制。我们的假设是，移植的FLSPC相对于宿主肝细胞的竞争优势来自于这些细胞中抗凋亡基因的过度表达，以及通过外源性细胞死亡途径促进宿主肝细胞的凋亡。我们将通过激光捕获显微切割从活跃的移植细胞团和周围宿主肝脏中提取RNA，并通过RT-PCR、qRT-PCR和高密度基因表达芯片研究特定基因的表达，从而确定参与其中的特定基因和信号通路；4)直接展示胎肝上皮祖细胞在体内的关键干细胞特性，包括克隆生长能力、双能性和自我更新能力。这些研究的总体目标是找到通过移植肝来源细胞来促进肝脏再繁殖的方法，最终将导致临床应用。公共卫生相关性：该项目旨在优化成年大鼠肝脏与胎肝干细胞/祖细胞的再繁殖，并确定调节这一过程的关键步骤、机制和参与细胞竞争的基因。

DESCRIPTION (provided by applicant): Currently, whole liver transplantation is the primary method used to treat end stage chronic liver failure. With the marked shortage of available donor livers, there has been considerable interest in identifying stem or progenitor cells can restore liver mass and function. We have demonstrated that fetal liver epithelial cells can repopulate up to 30% of parenchymal mass in normal liver, produce new hepatocytes and bile ducts and continue to proliferate for up to one year after cell transplantation. These are all properties of stem cells and we refer to these cells as fetal liver stem/progenitor cells (FLSPC). We hypothesize that use of a normal liver based cell transplantation system, such as the one we have established, is critical in determining the stem cell potential of isolated cells and cell lines and in identifying essential factors that contribute to their proliferation and differentiation in the liver. Recently, we discovered that fetal liver cells replace host hepatocytes by cell competition, a process originally described in Drosophila. Within this context, experiments are proposed: 1. To study the hypothesis that engraftment of fetal liver stem/progenitor cells into the hepatic parenchyma represents a rate limiting step in liver repopulation. These studies will utilize mouse and rat fetal liver stem/progenitor cells that have been purified to 95% homogeneity using immunomagnetic beads (MACS). 2. To study the hypothesis that modulating cell competition and understanding the circumstances under which this occurs will lead to identifying optimal conditions for liver repopulation by transplanted FLSPC. How long does repopulation/competition continue? Does the age of the host recipient at the time of cell transplantation influence the extent of repopulation? Is competition always driven by increased apoptosis in the host liver? and Can we block liver repopulation by knocking down expression of the anti-apoptotic gene, survivin, in FLSPC prior to their transplantation? 3. To identify specific genes, signaling pathways and mechanisms that regulate cell competition during mammalian liver repopulation. Our hypothesis is that the competitive advantage of transplanted FLSPC over host hepatocytes derives from overexpression of anti-apoptotic genes in these cells, coupled with augmented apoptosis of host hepatocytes through the extrinsic cell death pathway. We will identify the specific genes and signaling pathways involved by isolating RNA from actively proliferating transplanted cell clusters and surrounding host liver by laser-capture micro dissection and studying specific gene expression by RT-PCR, qRT-PCR and high density gene expression microarrays and 4) To directly demonstrate critical stem cell properties of fetal liver epithelial progenitor cells in vivo, including their ability to grow clonally, their bipotency and their capacity for self-renewal. The overall goal of these studies is to find methods to enhance liver repopulation by transplanted hepatic derived cells that will ultimately lead to clinical application in humans. Public Health Relevance: This project is directed toward optimizing repopulation of the adult rat liver with fetal liver stem/progenitor cells and in determining the critical steps, mechanism and genes involved in cell competition, which regulates this process.