ROLE OF ERYTHROPOIETIN IN TRANSCRIPTIONAL CONTROL

促红细胞生成素在转录控制中的作用

• 批准号: 3483167
• 负责人: Jerry L Spivak
• 金额: $ 20.18万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-04-01 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3483167
• 关键词: SDS polyacrylamide gel electrophoresis; acetaldehyde; blood /lymphatic neoplasm; cell differentiation; cell growth regulation; cell membrane; chelating agents; chromatography; digital imaging; divalent cations; erythropoiesis; erythropoietin; ethanol; hematopoietic stem cells; human tissue; hybridomas; laboratory mouse; membrane permeability; membrane structure; membrane transport proteins; myeloproliferative neoplasm; tissue /cell culture

The long term objective of this research project is to identify and characterize the molecular mechanisms involved in the regulation of eythropoiesis. To this end in the present proposal, the following investigations have been planned: First, we wish to characterize the intracellular mitogenic and transforming factors present in the peripheral blood mononuclear cells in polycythemia vera with respect to their role in the autonomous proliferation which characterizes this disorder. Using both conventional liquid chromatography and reversed phase-HPLC, as well as SDS-PAGE, we plan to isolate the growth factors we have previously identified for amino-terminal sequencing, amino acid composition and the production of synthetic peptides and antibodies to them. Using well defined assays for these growth factors, we will identify the cells in which they are expressed and whether they are secreted in the urine. We will also examine their ability to influence the proliferation of normal hematopoietic progenitor cells and the proliferation and function of marrow fibroblasts and vascular endothelial cells. The conditions influencing the production of these growth factors will be examined including the expression of particular proto-oncogenes by their cells of origin. Second, the fate and function of pure recombinant human erythropoietin will be studied in two different models. The metabolism of native and desialated erythropoietin will be studied in vivo in the rat and in vitro using rat hepatic parenchymal and endothelial cells as well as human and bovine vascular endothelial cells. The receptor-ligand interaction of erythropoietin and its target cells will be studied using erythroblasts isolated from the spleens of the phenylhydrazinetreated mice by centrifugal elutriation. In particular, we wish to examine the role of calcium in the interaction of erythropoietin and its target cells and will do this both with respect to receptor-ligand binding and subsequent intracellular events. The latter will be evaluated using a fluorescent intracellular calcium probe and a sensitive digital imaging system capable of analyzing changes in intracellular free calcium in single, living cells.

本研究项目的长期目标是确定和 表征参与调节的分子机制， 红细胞生成 为此目的，在本提案中， 调查已经计划：首先，我们希望描述 存在于外周血中的细胞内促有丝分裂和转化因子 真性红细胞增多症患者血单核细胞在 这种疾病的特征是自发的增殖。 同时使用 常规液相色谱法和反相HPLC法，以及 SDS-PAGE，我们计划分离出我们以前 确定了氨基末端测序、氨基酸组成和 合成肽及其抗体的生产。 使用公 这些生长因子的定义的测定，我们将确定细胞在 它们表达的方式以及它们是否在尿液中分泌。 我们 还将检查他们影响正常扩散的能力， 造血祖细胞与骨髓增殖和功能 成纤维细胞和血管内皮细胞。 影响的条件 这些生长因子的生产将被检查，包括 特定原癌基因在其来源细胞中的表达。 第二，纯重组人促红细胞生成素的命运和功能将 在两个不同的模型中进行研究。 天然的代谢和 将在大鼠体内和体外研究去唾液酸促红细胞生成素 使用大鼠肝实质和内皮细胞以及人和 牛血管内皮细胞 受体-配体相互作用 促红细胞生成素及其靶细胞将使用成红细胞进行研究 从苯肼处理的小鼠的脾脏中通过离心分离 淘洗 特别是，我们希望研究钙在 促红细胞生成素及其靶细胞的相互作用， 关于受体-配体结合和随后的细胞内 事件 后者将使用荧光细胞内 钙探针和灵敏的数字成像系统， 单个活细胞内游离钙的变化。