FUNCTIONAL STUDIES OF GRANULOCYTE MEMBRANES

粒细胞膜的功能研究

基本信息

批准号：
3229821
负责人：
Elizabeth R Simons
金额：
$ 17.43万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
1982
资助国家：
美国
起止时间：
1982-09-30 至 1992-08-31
项目状态：
已结题

项目摘要

Although the functional response of neutrophils, the attack on invading entitities by products of the oxidative burst and by the contents of the granules, has been studied in great detail, the mechanism by which this response follows upon exposure of the cells to their specific stimuli remains incompletely understood. It has been shown that, upon contact with soluble stimuli such as chemoattractants or phorbol esters, or with particulate agonists such as opsonized particles or immune complexes, granulocytes undergo rapid changes in membrane cation permeability, in cytoplasmic concentrations of Ca++, H+, Na+ and K+ ions, activation of an oxidative burst, release of granule contents and, for certain stimuli, enhanced phosphoinositide turnover and/or chemotaxis. The temporal sequence of these events, and their inter-dependence, as well as the nature of the transduction signals, remain unelucidated. While undifferentiated precursor cells appear unable to mount any of these responses except chemotaxis, their capability to do so rises as the cells mature into granulocytes (from human bone marrow myeloid precursor cell) or granulocytoids (from the human leukemic HL-60 cell line). The mechanism of the acquisition of this response capability is unknown. We plan to address these questions by examining neutrophils, as well as the precursor cells as a function of their stage of differentiation. We shall be concerned solely with the initial stages of the response (within the first 90 seconds) and will hence be investigating the kinetics of changes in the various cation gradients, in transmembrane potential, in intracellular Ca++ and H+, as a function of the stimulus and dose used, of the presence of specific cation channel blocking agents (such as dimethyl amiloride), of certain ionophores (such as valinomycin, ionomycin, nigericin), of functional blocking agents such as B. pertussis toxin, of the external Na+ and K+ concentrations (altered by altering the buffers), and of internal Ca++ (adjusted by using a cytoplasmic chelator such as BAPTA) and H+ (adjusted with NH4Cl or CH3COOH) concentrations. Because the reactions are rapid we shall used fluorescent indicators for most of the studies. Since there are advantages and disadvantages to each, we shall investigate not only the fluorescence changes in suspensions, which are averaged over the cell population, but also in single cells, as observed in a fluorescence activated cell sorter, which allows us to explore the possibility that our measurements reflect properties of only a portion of the cells.

尽管中性粒细胞的功能反应，但对氧化爆发的产物和颗粒的内容已经详细研究了这种反应在暴露后遵循的机制细胞到其特定的刺激尚不完全理解。已经表明，一旦与可溶性刺激接触化学吸引剂或佛波酯，或带有颗粒激动剂例如调向颗粒或免疫复合物，粒细胞经历膜阳离子渗透性的快速变化， Ca ++，H+，Na+和K+离子的细胞质浓度，氧化爆发的激活，颗粒含量的释放和，对于某些刺激，增强的磷酸肌醇周转和/或趋化性。这些事件的时间顺序，它们相互依赖性以及转导的性质信号，保持无效。而未分化的前体细胞似乎无法安装任何这些响应，除了趋化性，随着细胞的成熟，它们这样做的能力会上升进入粒细胞（来自人骨髓髓样前体细胞）或粒细胞（来自人类白血病HL-60细胞线）。收购此回应的机制功能是未知的。我们计划通过检查中性粒细胞以及前体细胞作为A 它们分化阶段的功能。我们将担心仅在响应的初始阶段（在第一个90中秒），因此将研究变化的动力学在各种阳离子梯度中，在跨膜电势中细胞内Ca ++和H+作为刺激和剂量的函数使用的是特定阳离子通道阻断剂的存在（例如二甲基二甲基），某些离子载体（例如功能阻断剂的瓣膜霉素，离子霉素，粘霉素）例如百日咳毒素，外部Na+和K+的浓度（通过更改缓冲区改变）和内部 Ca ++（使用胞质螯合剂（例如BAPTA）调整）和H+（用NH4CL或CH3COOH调整）浓度。由于反应很快，我们将使用荧光大多数研究的指标。由于有优势和每个人的缺点，我们不仅要调查悬浮液的荧光变化，在细胞群，但也在单细胞中，如在A中观察到的荧光激活的细胞分类器，这使我们能够探索我们的测量结果仅反映了细胞的一部分。