CULTURED HEPATOCYTES AS A MODEL OF ALCOHOL METABOLISM

在酒精性肝病动物模型的开发中， 很少有人注意到产生一种新的 使用原代培养的肝细胞的体外模型。 因此 本研究的总体目标是检查控制和调节 乙醇代谢的影响。 使用此 模型，我们建议测试：（1）延长培养期 与酒精接触有关。 目前，肝细胞可以 只在含酒精的培养基中培养7-9天， 而我们成功地将它们保持在无酒精环境中， 中等至1个月。 条件将被修改， 与含酒精培养基相关的时间， 几种方法：改变培养基，O2和CO2 可用性和细胞附着的基质。 (2)审查 通过这些调节乙醇和乙醛代谢 细胞培养7天以上。 研究的贡献 将评估三种已知的乙醇代谢途径 乙醇也有可能引发自身的 新陈代谢. 乙醇代谢的代谢后果， 脂肪肝的发生及对血清蛋白的影响 将评估合成和分泌。 调控 乙醛代谢和长期暴露于 乙醇对乙醇和乙醛脱氢酶活性的影响 研究了 (3)所有上述研究将使用 来自正常大鼠的肝细胞。 这些研究将得到 研究涉及长期酒精喂养大鼠的细胞， 乙醇的存在或不存在，以检查 耐受性的维持/消失。 (4)的诱导 和/或酒精P-450的维持。 除了乙醇 本身，我们将使用配体如吡唑、4-甲基吡唑或 异烟肼来维持或诱导酒精P-450。 细胞也将 从用这些试剂预处理的动物中培养。 (5)的 氧自由基和脂质过氧化在肝硬化中的作用 将评价与乙醇相关的肝毒性。 氧自由基的产生、脂质过氧化和对 长期暴露于乙醇后的氧化应激将 测定 预计对这些问题的审查 在精心控制的体外条件下， 为了更好地了解某些方面的发病机制， 长期酗酒导致的肝脏疾病

In the development of animal models of alcoholic liver disease, little attention has focused on the possibilities of generating an in vitro model using liver cells in primary culture. It is therefore the overall aim of this study to examine the control and regulation of ethanol metabolism in cultured rat hepatocytes. Using this model, we propose to test: (1) Extension of the culture period associated with ethanol exposure. At present, hepatocytes can only be cultured in alcohol containing medium for 7-9 days, whereas we have successfully maintained them in alcohol free medium for up to 1 month. Conditions will be modified to extend the period associated with alcohol containing culture medium in several ways: alterations to the culture medium, 02 and C02 availability, and substrate for cell attachment. (2) To examine the regulation of ethanol and acetaldehyde metabolism by these cells over at least 7 days culture. Studies on th contributions of the three known pathways of ethanol metabolism will be assessed as will be the possibility that ethanol can induce its own metabolism. Metabolic consequences of ethanol metabolism such as the generation of fatty liver and effect on serum protein synthesis and secretion will be evaluated. Regulation of acetaldehyde metabolism and the effect of prolonged exposure to ethanol on alcohol and aldehyde dehydrogenase activities will be investigated. (3) All of the above studies will be carried out using hepatocytes from normal rats. These studies will be paralleled by studies involving cells from chronic alcohol-fed rats cultured in the presence or absence of ethanol, to examine the maintenance/disappearance of tolerance. (4) The induction of and/or maintenance of the alcohol P-450. In addition to ethanol itself, we will use ligands such as pyrazole, 4-methylpyrazole or isoniazid to maintain or induce the alcohol P-450. Cells will also be cultured from animals pretreated with these agents. (5) The possible role of oxygen radicals and lipid peroxidation in the hepatotoxicity associated with ethanol will be evaluated. Generation of oxyradicals, lipid peroxidation and sensitivity to oxidative stress after prolonged exposure to ethanol will be determined. It is anticipated that examination of these parameters under carefully controlled conditions in vitro will lead to a better understanding of some aspects of the pathogenesis of liver disease following prolonged alcohol abuse.