QUANTITATIVE STUDIES ON GRANULOCYTE DIFFERENTIATION

这个项目的长期目标是更好地了解 末端蛋白的表达和调控机制 正常人与白血病人中性粒细胞的鉴别 粒细胞。急性髓系白血病(AML)与 在表达这种差异化的程序时出现混乱， 其临床特征是人口不断扩大 相对不成熟、功能不健全的细胞通常 表现出异常的颗粒生成。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打包成一类专门的 称为天青颗粒(AG)的溶酶体。AG展品 相当大的异质性似乎反映了这两个 成熟和功能上的差异。我们的具体目标是 旨在通过关联进一步定义AG异质性 超微结构分离颗粒的生化性质 细胞形态及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，我们到目前为止已经 确定了四种类型的AG，每种类型都可以归类为 不成熟或成熟。这种颗粒分类，连同自动化的 大小-频率颗粒直方图，将被用来分类 来自髓系白血病患者的颗粒。我们计划检查 某些亲天青的可能的过氧体性质 以及MPO和过氧化氢酶是否共定位于 嗜酸性粒细胞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、 酶活性的获得及MPO组装成 使用脉冲追逐程序的不同二聚体形式，自动化， 高效液相色谱、免疫沉淀、十二烷基硫酸钠- PAGE/荧光法和闪烁计数法分离， 鉴定和量化特定的分子物种。这些研究 将与识别和描述 运输和包装中涉及的胞内隔间 使用Percoll密度的组合将MPO引入不同的AG 梯度和免疫选择性分离技术。我们会 在生物化学领域将这些分子和细胞事件联系起来 和超微结构水平，并作为颗粒成熟的函数。 该项目将提供有关基本的 中性粒细胞分化机制应该是潜在的 对髓系白血病的诊断和分型有帮助 在设计和/或选择不同的治疗策略时 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.