ISOLATION AND ACTION OF ADULT RAT HEPATOCYTE MITOGENS

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 蓝色染料/HPLC）HGF将用于产生小鼠杂交瘤细胞系 其液体将通过标准的抗HGF活性筛选 酶联免疫吸附测定和ARL生物测定。 抗HGF-MCA 将获得的用于免疫亲和纯化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. 假设HGF和其他肽有丝分裂原（EGF，胰岛素和 胰高血糖素）激活生长所必需单价阳离子流 将用一组新的9只小鼠抗大鼠（Na+， 在本实验室构建了一个钾离子-ATP酶“泵”MCA。 首先是 “9 B1”（与胆小管膜结合的新MCA之一，和 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 充分表征的MCA，将传播到其他实验室 工作无关的问题），以确定是否（从ARL 和剂量反应生物测定），当（从间隔/持续时间和 增殖动力学研究），以及如何（从测量 哇巴因敏感ATP酶、~（86）Rb ~+内流和Na ~+、K ~+含量; 电镜免疫细胞化学定位研究） 化学诱导下MCA对肝细胞增殖转化的影响 定义条件。 这项研究将提供新的见解的基本问题， 有丝分裂原控制动物细胞增殖。 特别是，这项工作将 导致分离血液传播因子的新方法， 生长因子的作用与最终的 DNA合成的起始;以及关于结构的新信息， 肝钠泵的功能和位置。

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.