GENE EXPRESSION IN MATURE NEUTROPHILS

描述（改编自申请人的摘要）：中性粒细胞提供了对抗感染的基本防御，并且在由炎症引起的组织损伤中起主要作用。它们还为研究正常哺乳动物细胞中mRNA水平的调节提供了极好的简化模型。随着包含大量基因和表达序列标签的公共数据库的出现，人类遗传学的研究正在从单个基因的研究转向基因表达的全局分析。主要研究人员建议将这一重要的新方法应用于人类中性粒细胞。研究人员将使用基于cDNA 3'末端片段展示的方法，通过特异性限制酶产生，半定量地评估在活化的中性粒细胞中发现的大多数mRNA的水平。拟议的研究将测试的核心假设，即中性粒细胞显示复杂的，刺激特异性模式的遗传调控反应，这可以解释，至少部分地，通过组合使用“子程序”的协调调节反应。具体目标是：1）确定由多种明确的激动剂刺激的嗜中性粒细胞的基因表达模式，以及鉴定参与嗜中性粒细胞吞噬反应的新基因; 2）在体外和体内比较对简单激动剂与复杂生理激活剂（包括细菌和无菌炎症刺激）的反应模式; 3）通过抑制特异性受体和信号转导通路、暴露于临床上重要的抗炎药物以及确定复杂反应的时间和功能顺序，剖析对细菌和无菌炎症刺激的反应的组分; 4）确定基因转录水平变化的分子机制，包括转录控制机制和mRNA稳定性的调节。这些研究可以提供对已知和新基因的遗传库的洞察，这些基因有助于中性粒细胞反应，从而为增强宿主防御或改善炎症提供干预靶点。深入了解该系统中基因表达的转录和转录后机制也可能适用于其他更复杂的细胞类型和组织。

DESCRIPTION (Adapted from Applicant's Abstract): Neutrophils provide an essential defense against infection, and play a major role in the tissue damage caused by inflammation. They also provide an excellent, simplified model for studying the modulation of mRNA levels in normal mammalian cells. With the emergence of public databases containing very large numbers of genes and expressed sequence tags, research in human genetics is now turning from investigation of single genes to the global analysis of gene expression. The principal investigators propose to apply this important new approach to human neutrophils. Using methods based on display of cDNA 3' end fragments, generated by specific restriction enzymes, the investigators will semi-quantitatively assess the levels of most mRNAs found in activated neutrophils. The proposed studies will test the central hypothesis that neutrophils show complex, stimulus-specific patterns of genetic regulatory responses, which can be explained, at least in part, by combinatorial use of "subroutines" of coordinately regulated responses. The specific aims are: 1) Determine the pattern of gene expression by neutrophils stimulated by a wide variety of well- defined agonists, as well as identify new genes involved in the neutrophil phagocytic response; 2) Compare the patterns of response to simple agonists versus complex physiological activators, including bacterial and sterile inflammatory stimuli, both in vitro and in vivo; 3) Dissect the components of the responses to bacterial and sterile inflammatory stimuli by inhibition of specific receptors and signal transduction pathways, exposure to clinically important anti-inflammatory drugs, and determination of the temporal and functional order of the complex response; 4) Determine the molecular mechanisms underlying the changes in gene transcript levels, including both transcriptional control mechanisms and regulation of mRNA stability. These studies may provide insight into the genetic repertoire of known and new genes that contribute to the neutrophil response and hence provide targets for intervention in augmentation of host defense or amelioration of inflammation. Insight into transcriptional and post-transcriptional mechanisms of gene expression in this system may also be applicable to other, more complex, cell types and tissues.