ISOLATION AND ACTION OF ADULT HEPATOCYTE MITOGENS

成人肝细胞促分裂剂的分离和作用

• 批准号: 3228671
• 负责人: HYAM L LEFFERT
• 金额: $ 24.39万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-12-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3228671
• 关键词: adenosinetriphosphatase; autoradiography; cell growth regulation; coagulation factor X; electrofocusing; enzyme linked immunosorbent assay; epidermal growth factor; gel filtration chromatography; gene expression; growth factor receptors; high performance liquid chromatography; hybridomas; immunoprecipitation; ion transport; laboratory mouse; laboratory rabbit; laboratory rat; liver cells; liver regeneration; mature animal; mitogens; monoclonal antibody; nucleic acid hybridization; protooncogene; serum; tissue /cell culture; ultracentrifugation

The long term objective of this research is to understand how blood-borne substances control liver regeneration. To accomplish this goal, hepatic mitogens will be isolated and purified, characterized structurally, and analyzed for their mechanisms of action. Two hypotheses will be tested: (1) Rat plasma Factor X, a coagulation zymogen with a putative homology to the primary sequence of epidermal growth factor ("EGF"), stimulates hepatocyte proliferation by binding to specific hepatic cell surface receptors; and (2) Factor X and/or EGF receptors mediate sodium ion-dependent prereplicative events (proto-oncogene c-fos activation) required for proliferative transitions. Factor X will be purified from rat plasma (by ion exchange and/or immunoaffinity chromatography (using an anti-X mouse monoclonal antibody ("MCA") selected for its ability to block the Factor Xo Xa conversion)), characterized structurally (subunit M/r (SDS-PAGE); peptide maps (cellulose TLC); primary sequence (deduced from rat liver cDNA sequences cloned and detected with anti-X-MCA in the expression vector wavelength gtll)), and bioassayed for its mitogenic properties (using validated primary cultures of adult rat hepatocytes in the presence or absence of other known mitogens). Factor X receptors will be identified kinetically by monitoring the binding of 125 I-labelled Factor X to plasma membranes (or membrane fractions) from liver tissue and hepatocytes isolated or cultured under varying conditions. Kinetic binding constants (k1, k2, k/d, b/max) will be determined from non-equilibrium rate studies and Scatchard plots. The ability of Factor X to stimulate fos expression (increases in fos mRNA levels (detected by hybridization of 32P-v-fos probes on Northern blots), fos protein synthesis (detected by immunoprecipitation of 35S-met-p55 fos using a new c-fos-specific MCA), and %fos-producing hepatocytes (dual immunocytochemical detection with anti-fos MCA and anti- albumin antibody) will be tested in cultured hepatocyte bioassays. The prereplicative kinetics of fos expression--and its dependence on extracellular sodium ions--will be determined precisely and compared to fos activation responses obtained with EGF (in the presence or absence of other growth factors). Anti-fos MCA or anti-sense fos mRNA will be inserted into hepatocytes by rbc ghost/needle microinjection or by retroviral gene transduction, respectively, in attempts to block mitogen-activated fos expression (or function) and subsequent hepatocyte growth. Results from these studies will provide fundamental insights into the mechanisms that control and link hepatic growth and function.

这项研究的长期目标是了解如何 血液传播的物质控制肝脏再生。 完成 为此，将分离和纯化肝有丝分裂原， 结构特征，并分析其作用机制， 行动上 将检验两个假设：（1）大鼠血浆因子X， 一种凝血酶原，假定与主要的 表皮生长因子（EGF），刺激 特异性肝细胞结合促进肝细胞增殖 （2）因子X和/或EGF受体介导 钠离子依赖性复制前事件（原癌基因c-fos 激活）增殖性转变所需的。 因子X将是 从大鼠血浆中纯化（通过离子交换和/或免疫亲和 层析（使用抗X小鼠单克隆抗体 （“MCA”），其因其阻断因子Xo Xa的能力而被选择。 转化）），结构上表征（亚基M/r（SDS-PAGE）; 肽图（纤维素TLC）;一级序列（从大鼠 肝cDNA序列克隆，并与抗X-MCA检测， 表达载体波长λ 1），并对其进行生物测定 促有丝分裂特性（使用成年大鼠的经验证的原代培养物 在存在或不存在其它已知促分裂原的情况下的肝细胞）。 将通过监测X因子受体的活性来动力学鉴定X因子受体 125 I标记的因子X与质膜的结合（或 膜组分），或 在不同条件下培养。 动力学结合常数（k1， k2、k/d、B/max）将由非平衡速率确定 研究和Scatchard图。 X因子刺激 fos表达（fos mRNA水平增加（通过 ~（32）P-v-fos探针与fos蛋白（北方印迹）杂交 合成（通过35 S-met-p55 fos的免疫沉淀检测 使用新的c-fos特异性MCA）和% fos产生肝细胞 (dual用抗fos MCA和抗 白蛋白抗体）将在培养的肝细胞生物测定中进行测试。 Fos表达的复制前动力学及其依赖性 对细胞外钠离子的影响--将被精确地测定， 与用EGF获得的Fos激活反应相比（在 存在或不存在其它生长因子）。 抗Fos MCA或 反义fos mRNA通过红细胞插入肝细胞 血影/针显微注射或通过逆转录病毒基因转导， 分别尝试阻断丝裂原激活的FOS 表达（或功能）和随后的肝细胞生长。 这些研究的结果将提供基本的见解， 控制和连接肝脏生长和功能的机制。