HUMAN MAST CELL CHYMASE AND CATHEPSIN G EXPRESSION IN AIRWAY INFLAMMATION

这里提出的研究将比较主体的表达 正常人和炎症组织中人肥大细胞的胰凝乳酶样酶 并将确定遗传和核素的特征 对它们的调控表达负责。这些酶、凝乳酶和 组织蛋白酶G是MC/TC分泌颗粒的主要成分 肥大细胞的子集。它们有很强的促分泌剂、多肽酶和 基质降解活性，并释放到细胞外环境中 在抗原结合的IgE、神经肽和 其他刺激物。肥大细胞中含有凝乳酶和 组织蛋白酶G在不同组织中表现出显著的生理变化 肺和呼吸道的微环境及其分布模式 以及假设在肺和呼吸道中不同的产生水平 疾病。这个项目将探索酶的调节在 基因的水平。凝乳酶和细胞来源的水平、位置和细胞来源 组织蛋白酶G在正常和炎症呼吸道中的表达将被研究 组织，用定量聚合酶链式反应和原位 杂交。位于14号染色体两侧的顺式作用元件 这两种酶紧密相连的基因将通过转基因来确定。 含蛋白水解酶表达载体的凝乳酶转录细胞系 与记者基因相关的调控元件。特定DNA序列 参与与肥大细胞特异性核调节蛋白的结合将 由DNA足迹、凝胶迁移和甲基化来定义 干扰分析。最后，与肥大细胞有关的调节蛋白 乳糜酶和组织蛋白酶G的表达本身将被分离并 特色化的。这些研究涉及的基本问题是 具有重要生物学意义的肥大细胞类凝乳酶的表达 酶，并可能提供对鲜为人知的现象的见解 表型变异 在人肺的肥大细胞中。

The studies proposed here will compare the expression of the principal chymotrypsin-like proteases of human mast cells in normal and inflamed airway tissues and will characterize genetic and nuclear elements responsible for their regulated expression. These enzymes, chymase and cathepsin G, are major components of secretory granules in the MC/TC subset of mast cells. They have potent secretagogue, peptidase and matrix-degrading activity and are released into the extracellular milieu following activation of mast cells by antigen-bound IgE, neuropeptides and other stimuli. The proportion of mast cells that contains chymase and cathepsin G exhibits striking physiological variation in different tissue microenvironments of the lung and airways, with patterns of distribution and levels of production that are hypothesized to vary in lung and airway diseases. This project will explore the regulation of the enzymes at the level of the gene. The level, location and cellular source of chymase and cathepsin G mRNA expression will be explored in normal and inflamed airway tissues, using the quantitative polymerase chain reaction and in situ hybridization. The cis-acting elements on chromosome 14 flanking the closely linked genes for the two enzymes will be defined by transfection of chymase-transcribing cell lines with plasmids containing protease regulatory elements linked to reporter genes. Specific DNA sequences involved in binding to mast cell-specific nuclear regulatory proteins will be defined by DNA footprinting, gel mobility shift, and methylation interference assays. Finally, regulatory proteins involved in mast cell chymase and cathepsin G expression themselves will be isolated and characterized. These studies address basic questions regarding the expression of the biologically important mast cell chymotrypsin-like enzymes and may provide insights into the poorly understood phenomenon of phenotype variation in mast cells of human lung.