QUANTITATIVE STUDIES ON GRANULOCYTE DIFFERENTIATION

粒细胞分化的定量研究

基本信息

批准号：
2087029
负责人：
JOSEPH M KINKADE
金额：
$ 17.04万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-02-01 至 1995-07-31
项目状态：
已结题

项目摘要

The long-term objective of this project is to better understand the mechanisms underlying the expression and regulation of terminal differentiation of normal and leukemic human neutrophilic granulocytes. Acute myeloid leukemia (AML) is associated with a disorganization in the expression of this differentiative program, and is characterized clinically by an expanding population of relatively immature, functionally incompetent cells that often exhibit abnormal granulogenesis. Seventy percent of AML patients die of infections. Our experimental approach focuses on the enzyme myeloper-oxidase (MPO) which plays a major role in the microbicidal system of mature, functional neutrophils. We are studying the differentiation-stage synthesis, post-translational processing and packaging of different forms of MPO into a class of specialized lysomsomes known as azurophilic granules (AG). AG exhibit considerable heterogeneity that appears to reflect both maturational and functional differences. Our specific aims are directed at further defining AG heterogeneity by correlating biochemical properties of isolated granules with ultrastructural morphology and cytochemical and immunocytochemical staining of intact cells and granules. Using these criteria, we have so far identified four types of AG, each of which can be classed as immature of mature. Such granule typing, together with automated size-frequency granule histograms, will be used to classify the granules from patients with myeloid leukemia. We plan to examine the possible peroxisomal nature of certain azurophilic microgranules and whether MPO and catalase are co-localized as in the specific granules of eosinophils. We will study the relationship between the processing of MPO, the addition of heme, the acquisition of enzymatic activity and the assembly of MPO into different dimeric forms using pulse-chase procedures, automated, high resolution liquid chromatography, immunoprecipitation, SDS- PAGE/fluorography and scintillation counting methods to separate, identify and quantitate specific molecular species. These studies will be coordinated with efforts to identify and characterize the intracellular compartments involved in the transport and packaging of MPO into different AG using a combination of Percoll density gradient and immunoselective separation techniques. We will correlate these molecular and cellular events at the biochemical and ultrastructural levels and as a function of granule maturation. This project will provide new information concerning the basic mechanisms of neutrophil differentiation that should be potentially useful in the diagnosis and classification of myeloid leukemia and in the design and/or selection of different therapeutic strategies for the clinical management of leukemic patients.

该项目的长期目标是更好地了解终末表达和调控的机制正常人和白血病人中性粒细胞的区分粒细胞。急性髓系白血病 (AML) 与这种差异化程序的表达混乱，其临床特征是人群不断扩大相对不成熟、功能不健全的细胞，通常表现出异常的颗粒发生。 70% 的 AML 患者死于感染。我们的实验方法侧重于酶髓过氧化物酶（MPO）在杀菌中起主要作用成熟的、有功能的中性粒细胞系统。我们正在研究分化阶段合成、翻译后加工和将不同形式的 MPO 封装成一类专门的溶酶体称为嗜天青颗粒（AG）。 AG展品相当大的异质性似乎反映了两者成熟度和功能上的差异。我们的具体目标是旨在通过关联进一步定义 AG 异质性具有超微结构的分离颗粒的生化特性形态学、细胞化学和免疫细胞化学染色完整的细胞和颗粒。使用这些标准，到目前为止我们已经确定了四种类型的AG，每一种都可以分为成熟中的不成熟。这种颗粒分型以及自动化尺寸频率颗粒直方图，将用于对来自骨髓性白血病患者的颗粒。我们计划检查某些嗜天青的可能的过氧化物酶体性质微粒以及 MPO 和过氧化氢酶是否共定位，如嗜酸性粒细胞的特殊颗粒。我们将研究 MPO 加工、血红素添加之间的关系酶活性的获得和 MPO 的组装使用脉冲追踪程序的不同二聚体形式，自动化，高分辨液相色谱、免疫沉淀、SDS- PAGE/荧光照相术和闪烁计数方法来分离、识别和定量特定的分子种类。这些研究将与确定和描述参与运输和包装的细胞内区室使用 Percoll 密度组合将 MPO 引入不同的 AG 梯度和免疫选择性分离技术。我们将将这些分子和细胞事件与生化相关和超微结构水平以及颗粒成熟的函数。该项目将提供有关基本信息的新信息中性粒细胞分化的机制可能是有助于粒细胞白血病的诊断和分类不同治疗策略的设计和/或选择用于白血病患者的临床管理。