ISOLATION AND ACTION OF ADULT RAT HEPATOCYTE MITOGENS

成年大鼠肝细胞有丝分裂原的分离和作用

• 批准号: 3228669
• 负责人: HYAM L LEFFERT
• 金额: $ 20.37万
• 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 1987-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3228669
• 关键词: adenosinetriphosphatase; autoradiography; blood proteins; cell growth regulation; electrofocusing; enzyme linked immunosorbent assay; gel filtration chromatography; growth factor; high performance liquid chromatography; hybridomas; ion transport; liver cells; liver regeneration; mature animal; monoclonal antibody; serum; tissue /cell culture; ultracentrifugation

Monoclonal antibodies (MCA) and primary adult rat liver cell cultures (ARL) will be used to isolate a new hepatocyte serum protein growth factor (HGF) and to study its mechanism of action. Either partially (5700-fold) or further purified (cation exchange/Cibacron Blue Dye/HPLC) HGF will be used to generate mouse hybridoma cell lines whose fluids will be screened for anti-HGF activity by standard enzyme-linked immunoadsorbant assays and by ARL bioassays. Anti-HGF-MCA obtained will be used to immunoaffinity purify HGF, which will then be characterized by conventional and advanced semi-micro protein chemistry technology. The hypothesis that HGF and other peptide mitogens (EGF, insulin and glucagon) activate monovalent cation fluxes which are necessary for growth will be tested critically with a new panel of 9 mouse anti-rat (Na+, K+)-ATPase "pump" MCA's constructed in this laboratory. Starting with "9B1" (one of the new MCA's that binds to bile canalicular membranes, and inhibits ouabain-sensitive [Na+, K+]-ATPase as well as cultured hepatocyte DNA synthesis), detailed experiments (requiring large amounts of pure well-characterized MCA, that will be disseminated to other laboratories working on unrelated problems) will be conducted to determine if (from ARL and dose-response bioassays), when (from interval/duration and proliferation kinetic studies), and how (from measurements of ouabain-sensitive ATPase, 86Rb+ influx and Na+ and K+ content; and from electron microscopic immunocytochemical localization studies) anti-pump MCA's influence hepatocyte proliferative transitions under chemically defined conditions. This research will provide new insights into fundamental problems of how mitogens control animal cell proliferation. In particular, this work will lead to new approaches towards isolating blood borne factors that might relationships between the effects of growth factors and the eventual initiation of DNA synthesis; and to new information about the structure, function and location of the liver sodium pump.

单克隆抗体 (MCA) 和原代成年大鼠肝细胞培养物 (ARL) 将用于分离新的肝细胞血清蛋白生长因子（HGF） 并研究其作用机制。 部分纯化（5700 倍）或进一步纯化（阳离子交换/Cibacron Blue Dye/HPLC) HGF 将用于生成小鼠杂交瘤细胞系 其体液将按标准筛选抗 HGF 活性 酶联免疫吸附测定和 ARL 生物测定。 抗HGF-MCA 获得的产物将用于免疫亲和纯化HGF，然后将其 以传统和先进的半微量蛋白质化学为特征 技术。 HGF 和其他肽有丝分裂原（EGF、胰岛素和 胰高血糖素）激活生长所需的单价阳离子通量 将使用 9 种小鼠抗大鼠（Na+、 K+)-ATP 酶“泵”MCA 是在本实验室构建的。 开始于 “9B1”（与胆管膜结合的新 MCA 之一，以及 抑制哇巴因敏感的 [Na+, K+]-ATP 酶以及培养的肝细胞 DNA合成），详细的实验（需要大量的纯 特征明确的 MCA，将传播到其他实验室 将进行不相关问题的研究）以确定是否（来自 ARL 和剂量反应生物测定），何时（从间隔/持续时间和 增殖动力学研究），以及如何（通过测量 哇巴因敏感的 ATP 酶、86Rb+ 流入以及 Na+ 和 K+ 含量；和来自 电子显微镜免疫细胞化学定位研究）抗泵 MCA对化学条件下肝细胞增殖转变的影响 定义的条件。 这项研究将为解决如何解决这些问题的基本问题提供新的见解。 有丝分裂原控制动物细胞增殖。 特别是，这项工作将 导致分离血源性因子的新方法 生长因子的影响与最终结果之间的关系 DNA合成的起始；以及有关结构的新信息， 肝钠泵的功能和位置。