QUANTITATIVE STUDIES ON GRANULOCYTE DIFFERENTIATION

粒细胞分化的定量研究

• 批准号: 3165803
• 负责人: JOSEPH M KINKADE
• 金额: $ 13.4万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-08-01 至 1988-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3165803
• 关键词: autoradiography; blood fractionation; bone marrow exam; cell differentiation; density gradient ultracentrifugation; electron microscopy; gel electrophoresis; genetic translation; granulocyte; histochemistry /cytochemistry; immunochemistry; leukopoiesis; messenger RNA; molecular oncology; myelogenous leukemia; myeloid stem cell; myeloperoxidase; neutrophil; secretion; tissue /cell culture

The phenotypic expression of leukemia appears to be related to a disorganization in the normal processes of cellular commitment and differentiation within the hematopoietic system. Myeloid leukemic cells have been induced to undergo "normal" differentiation in vitro and in vivo. The resulting cells are no longer malignant in vivo and no longer multiply in vitro. The long-term objective of our studies is elucidation of the molecular mechanisms underlying the operation and regulation of normal and leukemic terminal myeloid differentiation with particular emphasis on the differentiation processes underlying granulogenesis. Our experimental approach has centered on the enzyme myeloperoxidase (MPO), an abundant, functionally important, granule-specific protein of neutrophilic polymorphonuclear leukocytes (PMN). MPO synthesis and incorporation into primary or azurophilic granules occurs exclusively during the promyelocyte stage of cytodifferentiation. We are studying these processes using the human promyelocytic leukemia cell line HL-60, which may be induced to differentiate in vitro to more mature PMN using agents such as dimethylsulfoxide. We have recently shown that PMN contains three forms of MPO that exhibit differences in enzymatic activity and subunit structure. There appears to be a differential distribution of these forms in different azurophilic granule subpopulations. Moreover, these different granule subpopulations appear to be under separate secretory control. We have developed a Percoll density gradient centrifugation system for the isolation of 13 granule subpopulations from normal human PMN. Biochemical and ultrastructural characterization of these subpopulations revealed striking qualitative and quantitative differences. We have isolated the mRNA for MPO from HL-60 cells and are studying translation and processing in vitro. We have developed a new cationic detergent-PAGE system for the determination of molecular weights of biologically active proteins including MPO. Current work is aimed at using these procedures to correlate the biosynthesis and packaging of MPO in leukemic cells at the cellular and molecular levels. We feel the information to be developed by this project has clear implications for the future development of new strategies for the clinical management of myeloid leukemia. (M)

白血病的表型表达似乎与 细胞承诺正常过程的混乱和 造血系统内的分化。 髓系白血病细胞 已被诱导在体外和体内进行“正常”分化 体内。 产生的细胞在体内不再是恶性的，也不再是 在体外繁殖。 我们研究的长期目标是阐明 的运作和调节的分子机制 正常和白血病终末髓系分化，具有特定的 强调颗粒发生的分化过程。 我们的 实验方法以髓过氧化物酶（MPO）为中心，这是一种 丰富的、功能重要的、中性粒细胞的颗粒特异性蛋白 多形核白细胞（PMN）。 MPO 合成并掺入 初级或嗜天青颗粒仅发生在早幼粒细胞期间 细胞分化阶段。 我们正在使用以下方法研究这些过程 人早幼粒细胞白血病细胞系 HL-60，可诱导 使用以下试剂在体外分化为更成熟的 PMN 二甲亚砜。 我们最近表明 PMN 包含三种形式 MPO 在酶活性和亚基结构方面表现出差异。 这些形式在不同地区似乎存在差异性分布 嗜天青颗粒亚群。 此外，这些不同的颗粒 亚群似乎处于单独的分泌控制之下。 我们有 开发了 Percoll 密度梯度离心系统 从正常人 PMN 中分离出 13 个颗粒亚群。 生化 这些亚群的超微结构特征揭示 质和量上的显着差异。 我们已经隔离了 来自 HL-60 细胞的 MPO mRNA，正在研究翻译和加工 体外。 我们开发了一种新型阳离子去污剂-PAGE系统 生物活性蛋白质分子量的测定 包括 MPO。 当前的工作旨在利用这些程序 关联白血病细胞中 MPO 的生物合成和包装 细胞和分子水平。 我们认为需要开发的信息 该项目对新产品的未来发展具有明显的影响 骨髓性白血病的临床治疗策略。 (男)