ISOLATION AND ACTION OF ADULT HEPATOCYTE MITOGENS

这项研究的长期目标是了解 血液传播的物质控制肝脏再生。要完成 为了达到这个目标，肝脏有丝分裂原将被分离和提纯， 对它们的结构进行了表征，并对它们的机理进行了分析 行动。将检验两个假设：(1)大鼠血浆因子X， 一种凝血酶原，推测与初级 表皮生长因子(EGF)序列，刺激 肝细胞与特异性肝细胞结合后的增殖 表面受体；和(2)因子X和/或EGF受体介导 钠离子依赖复制前事件(原癌基因c-fos 激活)是增殖转变所必需的。因子X将是 从大鼠血浆中提纯(通过离子交换和/或免疫亲和 层析(使用抗X鼠单抗 (“MCA”)因其阻止因子XO Xa的能力而被选中 转化))，结构特征(M/r亚基(SDS-PAGE)； 多肽图谱(纤维素层析)；一级序列(源自大鼠 大鼠肝脏c DNA序列的克隆及抗X-MCA抗体检测 表达载体波长gt11))，并对其进行生物检测 有丝分裂特性(使用经过验证的成年大鼠原代培养 肝细胞在存在或不存在其他已知有丝分裂原的情况下)。 凝血因子X受体将通过监测 125I标记的凝血因子X与质膜的结合(或 膜组分)从肝组织和分离的肝细胞 在不同的条件下培养。动力学结合常数(k1， K2、k/d、b/max)将由非平衡率确定 研究和斯卡查德的情节。凝血因子X的刺激能力 FOS表达(FOS mRNA水平的增加(通过 ~(32)P-v-FOS探针在Northern杂交中的应用)、FOS蛋白 合成(通过35S-MET-P55 FOS的免疫沉淀检测 使用一种新的c-fos特异性MCA)和%fos产生肝细胞 (抗FOS MCA和抗FOS MCA双重免疫细胞化学检测 白蛋白抗体)将在培养的肝细胞生物检测中进行检测。 Fos表达的复制前动力学--及其依赖性 在胞外钠离子上-将被精确地测定和 与使用EGF获得的FOS激活反应相比(在 存在或不存在其他增长因素)。防FOS MCA或 反义FOS mRNA将通过RBC插入肝细胞 幽灵/针头显微注射或逆转录病毒基因转导， 分别在试图阻断丝裂原激活的FOS的过程中 表达(或功能)和随后的肝细胞生长。 这些研究的结果将为我们提供对 控制和连接肝脏生长和功能的机制。

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.