GROWTH FACTOR CA++ SIGNALING IN ALCOHOLIC LIVER DISEASE

酒精性肝病中的生长因子 CA 信号传导

• 批准号: 3432685
• 负责人: JOHN R. WILLIAMSON
• 金额: $ 2.15万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3432685
• 关键词: alcoholic liver cirrhosis; biological signal transduction; biopsy; calcium; calcium indicator; growth factor; hormone regulation /control mechanism; human subject; liver cells; transforming growth factors

A research program is described which will be directed towards the characterization of growth factor-medicated Ca2+ responses in human parenchymal liver cells using liver biopsy material. This study relates to an extension of the parent grant concerning epidermal growth factor (EGF) and hepatocyte growth factor (HGF) actions on the liver. The physician experience of the Hungarian collaborator and his experience with measurement of growth factor effects on Ca2+ homeostasis in single cells will be combined to apply advanced techniques to human liver cells. First, attention will be focussed on establishing a reliable technique for measurement of intracellular Ca2+ in fura-2-loaded single human hepatocytes to characterize "normal" Ca2+ homeostasis of these cells with an emphasis on the effects of the recently described HGF. The involvement of a receptor tyrosine kinase in HGF-induced Ca2+ mobilization will be evaluated by tyrosine kinase inhibitors. The amount of releasable Ca2+ from non-mitochondrial and mitochondrial pools of human hepatocytes will be assessed by microperfusion of the specific Ca2+-mobilizing agents such as thapsigargin and mitochondrial uncouplers. Furthermore, purified antibodies raised against different components of the signaling pathway (G-proteins, phospholipase C isozymes) will be microinjected into human hepatocytes. This powerful approach has the potential for giving very specific answers to questions related to hormone-induced signal transduction in human liver. Second, growth factor-induced signal transduction and Ca2+ homeostasis will be investigated throughout the pathological evolution of alcoholic liver disease (ALD). We hypothesize that cellular changes in the hepatocytes are accompanied by specific alterations in cellular Ca2+ homeostasis corresponding in severity to the stage of ALD. A higher basal Ca2+ and a reduced cellular responsiveness to the effect of Ca2+ mobilizing hormones and growth factors is expected in relation to a decrease in the capacity of hormone-sensitive pools to sequester Ca2+. Assessment of the size of non-mitochondrial and mitochondrial Ca2+ pools will allow an evaluation of abnormal redistribution in the intracellular Ca2+. The inhibitory properties of transforming growth factor beta (TGF- beta) as well as the effect of protein kinase C-stimulating phorbol esters on HGF-induced Ca2+ response will be also determined. Knowledge of Ca2+ homeostasis and HGF-related early biochemical events of human parenchymal liver cells obtained throughout this project is of direct relevance to the development of new strategies for the prevention, prognostication, and treatment of alcoholic liver disease.

描述了一个研究计划，该计划将针对 生长因子对人体内钙离子反应的特性 肝实质细胞采用肝活检材料。这项研究与 关于表皮生长因子的父母拨款的延期 EGF和肝细胞生长因子(HGF)对肝脏的作用。这个 匈牙利合作者的医生经验和他的经验 测定生长因子对单个核细胞内钙稳态的影响 这些细胞将被组合在一起，将先进的技术应用于人类肝细胞。 首先，将把注意力集中在建立可靠的技术上 Fura-2负载的人细胞内钙离子浓度的测定 肝细胞表征这些细胞的“正常”钙稳态 强调最近描述的肝细胞生长因子的影响。这个 受体酪氨酸激酶参与肝细胞生长因子诱导的钙离子 动员将通过酪氨酸激酶抑制剂进行评估。这笔钱 非线粒体库和线粒体库中可释放的钙离子 人类肝细胞将通过特定的微灌注法进行评估 钙离子动员剂，如thapsigargin和线粒体解偶联剂。 此外，针对不同成分的纯化抗体 信号通路(G蛋白、磷脂酶C同工酶)将是 显微注射入人肝细胞。这种强大的方法具有 有可能对以下问题给出非常具体的答案 激素诱导的人体肝脏信号转导。 第二，生长因子诱导的信号转导与钙稳态 将在整个酒精中毒的病理演变过程中进行调查 肝病(ALD)。我们假设细胞在细胞内的变化 肝细胞伴随细胞内钙离子的特殊变化 动态平衡的严重程度与ALD的分期相对应。一个更高的 基础钙离子与细胞对钙离子影响的反应性降低 动员激素和生长因子预计与 激素敏感池隔离钙离子的能力下降。 非线粒体和线粒体钙池大小的评估 将允许评估细胞内的异常重新分布 Ca2+。转化生长因子-β的抑制作用 β)以及蛋白激酶C刺激佛波醇的作用 此外，还将测定酯类对HGF诱导的钙反应的影响。 钙稳态与肝细胞生长因子相关早期生化事件的认识 在整个项目中获得的人肝实质细胞的比例是 与制定新的预防战略直接相关， 酒精性肝病的预测和治疗。