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 血源性物质控制肝脏再生。 为了完成 为了这个目标，肝丝裂原将被分离和纯化， 对其结构进行表征，并分析其作用机制 行动。 将测试两个假设：(1) 大鼠血浆因子 X， 与初级具有假定同源性的凝固酶原 表皮生长因子（“EGF”）序列，刺激 通过与特定肝细胞结合而促进肝细胞增殖 表面受体； (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）是增殖转变所必需的。 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”）因其阻断 Xo Xa 因子的能力而被选中 转化）），结构表征（亚基 M/r (SDS-PAGE)； 肽图（纤维素薄层色谱）；一级序列（从大鼠推导 肝脏 cDNA 序列克隆并用抗 X-MCA 检测 表达载体波长gtll))，并对其进行生物测定 有丝分裂特性（使用经过验证的成年大鼠原代培养物 存在或不存在其他已知有丝分裂原的肝细胞）。 X 因子受体将通过监测 125 I标记的因子X与质膜的结合（或 膜部分）从肝组织和肝细胞分离或 在不同条件下培养。 动力学结合常数 (k1, k2、k/d、b/max) 将根据非平衡率确定 研究和斯卡查德图。 X因子的刺激能力 fos 表达（fos mRNA 水平增加（通过检测） 32P-v-fos 探针在 Northern 印迹上的杂交），fos 蛋白 合成（通过 35S-met-p55 fos 的免疫沉淀检测 使用新的 c-fos 特异性 MCA）和 %fos 生成肝细胞 （使用抗-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 细胞外钠离子——将被精确测定 与 EGF 获得的 fos 激活反应相比（在 是否存在其他生长因子）。 Anti-fos MCA 或 反义fos mRNA将被红细胞插入肝细胞 幽灵/针显微注射或通过逆转录病毒基因转导， 分别试图阻止丝裂原激活的 fos 表达（或功能）和随后的肝细胞生长。 这些研究的结果将为以下问题提供基本见解： 控制和连接肝脏生长和功能的机制。