Mechanisms of Polyploidy and Aneuploidy in the Liver

描述(申请人提供)：近2500万美国人受到肝功能障碍的影响，肝病是美国第十大死因。显然，迫切需要开发新的替代整个器官的替代方案。需要更好地了解肝脏生物学，以改进现有的方法，并创新治疗包括病毒性肝炎和脂肪性肝炎在内的肝病的治疗方法。肝细胞是肝脏的主要功能细胞类型，由于普遍存在的生理性多倍体(小鼠为90%，人类为50%)和非整倍体(小鼠为60%，人类为30%-90%)，肝细胞显示出一系列的染色体多样性。在真核生物中，细胞通常含有由一对同源染色体组成的二倍体基因组。多倍体是指整组染色体的获得，非整倍体是指单个染色体的获得和丢失。肝脏多倍体和非整倍体的作用代表了我们目前对肝脏生物学理解的一个主要差距。我们最近发现，非整倍体可以增强小鼠肝脏的再生能力。为了应对酪氨酸血症引起的损伤(通常对肝脏有毒性)，我们鉴定了一组对这种疾病具有抵抗力的非整倍体肝细胞。这些数据表明，非整倍体肝细胞具有更强的适应和再生能力。我们的中心假设是，非整倍体作为一种适应机制对肝损伤做出反应。这项应用的目标是确定调节肝脏非整倍体/多倍体的机制，并揭示非整倍体如何影响肝功能。为了研究这些问题，我们在特定的目标1中提出了确定多倍体肝细胞是否是非整倍体肝脏发育所必需的。实验将对E2f7/E2f8基因敲除小鼠的肝细胞分裂、核型和应激反应进行表征，这些小鼠具有正常的肝功能，但缺乏多倍体肝细胞。在特定的目标2中，我们将剖析最近在我们实验室发现的一种新的肝脏多倍体调节因子microRNA-122(miR-122)的作用。实验将确定miR-122如何改变一生中的倍性和非整倍体。我们还将确定miR-122调节肝脏倍体的细胞和分子机制。最后，在具体目标3中，我们将确定随机核型(在非整倍体肝细胞中)如何影响肝脏的功能。我们将利用一种新的异种移植模型来检查再生的人肝细胞的克隆性结节。实验将测量非整倍体，并确定这些结节中的基因表达谱。总之，这些研究将确定非整倍体对肝脏修复/再生的影响程度，以及控制这一过程的分子机制。了解非整倍体肝细胞是如何产生和发挥功能的，将为了解肝脏的动态平衡、疾病和治疗提供新的和关键的见解。

DESCRIPTION (provided by applicant): Nearly 25 million Americans are affected by liver dysfunction, and liver diseases are the 10th leading cause of death in the US. There is a clear and urgent need for developing new alternatives to whole organ replacement. A better understanding of liver biology is required to improve existing approaches and to innovate therapies for the treatment of liver diseases, including viral hepatitis and steatohepatitis. Hepatocytes, the primary functional cell type in the liver, display a range of chromosomal diversity resulting from prevalent physiological polyploidy (>90% in mice and 50% in humans) and aneuploidy (60% in mice and 30-90% in humans). In eukaryotic organisms, cells usually contain a diploid genome comprised of pairs of homologous chromosomes. Polyploidy refers to gains in entire sets of chromosomes, and aneuploidy refers to gains and losses of individual chromosomes. The roles of hepatic polyploidy and aneuploidy represent a major gap in our current understanding of liver biology. We recently found that aneuploidy enhances the regenerative capacity of the mouse liver. In response to Tyrosinemia-induced injury, that is normally toxic to the liver, we identified a subset of aneuploid hepatocytes that was resistant to the disease. The data suggest that aneuploid hepatocytes are endowed with enhanced capacity for adaptation and regeneration. Our central hypothesis is that aneuploidy functions as an adaptive mechanism in response to hepatic injury. The goals of this application are to identify mechanisms regulating hepatic aneuploidy/polyploidy and to unravel how aneuploidy affects liver function. To investigate these questions, we propose in Specific Aim 1 to determine whether polyploid hepatocytes are necessary for development of aneuploid livers. Experiments will characterize hepatic cell divisions, karyotypes and stress response in E2f7/E2f8 knockout mice, which have normal liver function but are depleted of polyploid hepatocytes. In Specific Aim 2, we will dissect the role of a novel regulator of hepatic polyploidy, recently identified in ur laboratory, microRNA-122 (miR-122). Experiments will determine how miR-122 alters ploidy and aneuploidy throughout life. We will also identify cellular and molecular mechanisms by which miR-122 regulates hepatic ploidy. Finally, in Specific Aim 3, we will determine how random karyotypes (in aneuploid hepatocytes) affect function in the liver. We will utilize a novel xenotransplantation model to examine clonal nodules of regenerating human hepatocytes. Experiments will measure aneuploidy and determine gene expression profiles in these nodules. Together, these studies will define the extent to which aneuploidy affects liver repair/regeneration as well as the molecular mechanisms that control this process. Understanding how aneuploid hepatocytes arise and function will provide new and crucial insights into liver homeostasis, diseases and treatments.