QUANTITATIVE STUDIES ON GRANULOCYTE DIFFERENTIATION

该项目的长期目标是更好地了解 终末转录因子的表达和调节机制 正常人和白血病人嗜酸性粒细胞的鉴别 粒细胞 急性髓性白血病（AML）与 在表达这个分化程序时的混乱， 并且临床上的特征是 相对不成熟，功能不全的细胞， 表现出异常的颗粒形成。 70%的AML患者 死于感染 我们的实验方法集中在酶 髓过氧化物酶（MPO）在杀微生物剂中起主要作用， 系统的成熟，功能性中性粒细胞。 我们正在研究 分化阶段的合成，翻译后加工和 将不同形式的MPO包装成一类专门的 溶体称为嗜天青颗粒（AG）。 AG展品 相当大的异质性，似乎反映了这两个 成熟和功能的差异。 我们的具体目标是 旨在通过关联以下因素来进一步定义AG异质性： 具有超微结构的分离颗粒的生化性质 形态学和细胞化学和免疫细胞化学染色 完整的细胞和颗粒。 根据这些标准，到目前为止， 确定了四种类型的AG，每一种都可以归类为 不成熟的成熟。 这种颗粒分型，连同自动化的 大小频率颗粒直方图，将用于分类 粒细胞白血病患者的骨髓中。 我们计划检查 某些嗜天青菌可能具有过氧化物酶体性质 微粒和MPO和过氧化氢酶是否共定位， 嗜酸性粒细胞的特殊颗粒。 我们会研究 MPO的加工，血红素的添加， 酶活性的获得和MPO组装成 不同的二聚体形式，使用脉冲追踪程序，自动化， 高分辨液相色谱，免疫沉淀，SDS- PAGE/荧光法和闪烁计数法分离， 鉴定和定量特定分子种类。 这些研究 将与确定和描述 参与运输和包装的细胞内区室 的MPO到不同的AG使用Percoll密度的组合， 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在生物化学上联系起来， 和超微结构水平以及作为颗粒成熟的函数。 该项目将提供有关基本 中性粒细胞分化的机制， 可用于骨髓性白血病的诊断和分类， 在不同治疗策略的设计和/或选择中 用于白血病患者的临床管理。

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.