GROWTH, DIFFERENTIATION, AND MATURATION OF MAST CELLS

组织肥大细胞的异质性现在在 小鼠、大鼠和人类，但对肥大细胞是如何发育的知之甚少 来自骨髓祖细胞还是有多么不同的表型和伴随 职能受到规范。细胞因子调控的体外研究进展 表型不同的小鼠肥大细胞群体， 编码蛋白多糖核心的基因的克隆和多只小鼠 肥大细胞蛋白水解酶(MMCP)和抗体的产生 为这些颗粒成分的具体分析提供了机会 基因转录调控、信使核糖核酸稳定性、蛋白质 蛋白水解酶的翻译、颗粒靶向和成熟 不同群体培养的小鼠肥大细胞。在特定目标中1 在这个项目中，细胞因子诱导 和/或抑制编码颗粒的mRNA的稳态水平 小鼠骨髓来源的肥大细胞(MBMMC)中的蛋白酶将被 学习。14号染色体复合体的甲基化模式 肥大细胞糜酶(mMCP-1至mMCP-5)基因驻留在 表达和不表达这些蛋白酶的细胞。DNA酶-I 超敏反应分析和瞬时转基因实验 利用人生长激素基因/mMCP报告基因构建 活性位点的删除和定点突变将用于 确定5‘端顺式作用元件中的关键核苷酸 MMCP-4和mMCP-7基因的侧翼区和/或内含子 在不同的肥大细胞群体中调节它们的转录。 肥大细胞特异性反式作用因子调节转录 这些MMCP将通过凝胶迁移率变化分析进行鉴定；然后他们 将被紫外线标记和提纯。一份核运行分析，随后是 一种类似于鉴定细胞的瞬时转染法 细胞因子转录本中富含Au的不稳定结构域将用于 识别mMCP转录本中调节 它们在MBMMC中的稳定性。在特定目标2中，免疫化学和 生化技术将用于动力学实验，以研究 某些细胞因子对翻译和翻译的调节作用 MBMMC瞬时转染过程中蛋白水解酶的颗粒积累 将在大鼠身上进行定点突变实验 嗜碱性白血病细胞鉴定特定氨基酸残基的研究 在控制其靶向颗粒及其相互作用mMCP-5中 含丝氨酸蛋白多糖。

The heterogeneity of tissue mast cells is now well recognized in the mouse, rat, and human, but little is known about how mast cells develop from bone marrow progenitors or how distinct phenotypes and attendant functions are regulated. The in vitro development of cytokine-regulated populations of mouse mast cells that are phenotypically distinct, the cloning of genes that encode the proteoglycan core and numerous mouse mast cell proteases (mMCP), and the production of antibodies that are specific to these granule constituents provide the opportunity to analyze the regulation of gene transcription, mRNA stability, protein translation, and granule targeting and maturation of proteases in different populations of cultured mouse mast cells. In Specific Aim 1 of this Project, the molecular mechanisms by which cytokines induce and/or suppress the steady-state levels of mRNAs that encode granule proteases in mouse bone marrow-derived mast cells (mBMMC) will be studied. The methylation pattern of the chromosome 14 complex where the mast cell chymase (mMCP-1 to mMCP-5) genes reside will be analyzed in cells that do and do not express these proteases. DNase-I hypersensitivity analyses and transient transfection experiments employing human growth hormone gene/mMCP reporter constructs with deletion and site-directed mutagenesis of active sites will be used to identify the critical nucleotides in the cis-acting elements in the 5' flanking regions and/or introns of the mMCP-4 and mMCP-7 genes that regulate their transcription in different populations of mast cells. Mast cell-specific trans-acting factors that regulate transcription of these mMCPs will be identified by gel-mobility-shift assays; they then will be UV -tagged and purified. A nuclear-run-on analysis followed by a transient-transfection approach analogous to that used to identify the AU-rich destabilizing domain in cytokine transcripts will be used to identify the specific nucleotides in the mMCP transcripts that regulate their stability in mBMMC. In Specific Aim 2, immunochemical and biochemical techniques will be used in kinetic experiments to investigate the effects of certain cytokines on the regulation of translation and granule accumulation of proteases in mBMMC Transient-transfection and site-directed mutagenesis experiments will be carried out in rat basophilic leukemia cells to identify the specific amino acid residues in mMCP-5 that control its targeting to the granule and its interaction with serglycin proteoglycans.