Growth Control of Normal and Cirrhotic Hepatocytes

描述(由申请人提供)： 肝细胞是一种独特的实质细胞，它能分裂和再生肝脏，以应对重大损伤。然而，这种再生能力会导致慢性损伤的肝硬变肝细胞生长紊乱。肝硬变肝脏中的肝细胞对生长控制信号的反应不同。大多数肝细胞癌发生在肝硬变的肝脏，这为肝硬变的肝细胞改变生长控制行为提供了证据。肝硬变是一个主要的世界性健康问题，由于酗酒和乙型和丙型肝炎病毒的传播，预计患病率将会增加，所有这些都是导致肝硬变的主要原因。在早期的提案(K08)中，我们假设肝硬变的肝细胞对细胞凋亡不那么敏感。事实上，研究证实，肝硬变肝细胞对多种凋亡剂的反应较差，包括肿瘤坏死因子α(TNFpha)、转化生长因子β(TGFbeta)和紫外线-C辐射(UV-C)。由于这种细胞因子是已知的肝细胞生长抑制因子，并且在肝硬变中的浓度很高，因此对转化生长因子β诱导的细胞凋亡的抵抗特别感兴趣。TGFR诱导肝细胞凋亡的机制尚不清楚，但我们在原代正常小鼠肝细胞中的研究表明，这是通过caspase-8依赖的机制发生的，该机制需要产生活性氧物种，需要细胞色素c释放导致线粒体去极化，并激活caspase-3。这是caspase-8依赖的第一个证明。在肝硬变肝细胞中抑制这一途径的机制尚不清楚，但可能涉及其他TGFR激活的途径，如Smad信号通路。我们假设Smad信号通路是肝细胞凋亡所必需的，并且在肝硬变的肝细胞中存在这一通路的缺陷。Smad信号通路的改变可能会改变控制细胞生存和死亡之间平衡的促或抗凋亡因子。在这个方案中，我们的目的是确定Smad信号转导通路是否在TGFβ诱导的肝细胞凋亡中是必要的。此外，由于肝硬变肝细胞表现出对凋亡的抵抗，这可能是由于促凋亡和抗凋亡因子的表达变化所致，我们假设正常肝细胞和肝硬变肝细胞的基因表达谱不同。我们建议鉴定和描述正常肝细胞和肝硬变肝细胞对凋亡刺激的反应基因表达谱的差异。对这些假设的检验将使我们的实验室在深度和广度上都有所增长，因为我们将使用北卡罗来纳大学基因组学核心和微阵列设施提供的新微阵列技术。我们将继续向我们的K08导师(David Brenner博士和Lola Reid博士)寻求建议，并免费使用北卡罗来纳大学胃肠道生物学和疾病中心的核心设施，如分子成像核心。

DESCRIPTION (provided by applicant): Hepatocytes are unique parenchymal cells that divide and regenerate the liver in response to major injury. This regenerative capacity, however, leads to disordered hepatocyte growth in the chronically injured, cirrhotic liver. Hepatocytes in the cirrhotic liver respond differently to growth control signals. Most hepatocellular carcinomas arise in a cirrhotic liver, providing evidence that cirrhotic hepatocytes have altered growth control behavior. Cirrhosis is a major worldwide health problem that is expected to increase in prevalence because of increasing alcohol abuse and dissemination of the hepatitis B and C viruses, all major causes of cirrhosis. In an earlier proposal (K08), we hypothesized that cirrhotic hepatocytes would be less sensitive to apoptosis. Indeed, studies confirmed that cirrhotic hepatocytes are less responsive to multiple apoptotic agents including tumor necrosis factor a (TNFalpha), transforming growth factor beta (TGFbeta), and ultraviolet-C irradiation (UV-C). The resistance to TGFbeta-induced apoptosis is of special interest because this cytokine is a known inhibitor of hepatocyte growth, and it is present in high concentrations in the cirrhotic liver. The mechanisms by which TGFR induces hepatocyte apoptosis are not well known, but our studies in primary normal mouse hepatocytes suggest that this occurs through a caspase-8 dependent mechanism that requires the generation of reactive oxygen species, depolarization of the mitochondria with cytochrome c release, and caspase-3 activation. This is the first demonstration of caspase-8 dependence. The mechanisms that inhibit this pathway in cirrhotic hepatocytes are unknown, but may involve other TGFR-activated pathways such as the Smad signaling pathway. We hypothesize that the Smad signaling pathway is necessary for hepatocyte apoptosis, and that defects in this pathway are present in cirrhotic hepatocytes. Alterations in the Smad signaling pathway may alter pro- or anti-apoptotic factors that govern the cellular balance between survival and death. In this proposal, we aim to determine if the Smad signal transduction pathway is necessary for TGFbeta-induced hepatocyte apoptosis. In addition, because cirrhotic hepatocytes exhibit resistance to apoptosis that may be due to changes in expression of pro-and anti-apoptotic factors, we hypothesize that normal and cirrhotic hepatocytes differ in their gene expression profiles. We propose to identify and characterize differences in gene expression profiles between normal and cirrhotic hepatocytes in response to apoptotic stimuli. Examination of these hypotheses will allow our laboratory to growth both in depth and in breadth, because we will use new microarray technology provided within the UNC Genomics Core and Microarray Facility. We will continue to seek counsel from our K08 mentors (Drs. David Brenner and Lola Reid) and use liberally the UNC Center for Gastrointestinal Biology and Disease for core facilities such as the Molecular Imaging Core.