GROWTH, DIFFERENTIATION, AND MATURATION OF MAST CELLS

组织肥大细胞的异质性现在已被广泛认识， 小鼠、大鼠和人类，但对肥大细胞如何发育知之甚少 从骨髓祖细胞或如何不同的表型和伴随 功能是规范的。 苦参碱调节的体外开发 表型不同的小鼠肥大细胞群体， 克隆编码蛋白聚糖核心的基因， 肥大细胞蛋白酶（mMCP），以及抗体的产生， 这些颗粒成分的特异性提供了分析的机会 基因转录、mRNA稳定性、蛋白质 翻译，颗粒靶向和成熟的蛋白酶， 不同群体的培养小鼠肥大细胞。 具体目标1 在本项目中，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 研究了 14号染色体复合体的甲基化模式， 肥大细胞糜蛋白酶（mMCP-1至mMCP-5）基因将在 表达和不表达这些蛋白酶的细胞。 DNA酶-Ⅰ 超敏性分析和瞬时转染实验 使用人生长激素基因/mMCP报告基因构建体， 活性位点的缺失和定点诱变将用于 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， 调节它们在不同肥大细胞群体中的转录。 肥大细胞特异性的反式作用因子，调节 这些mMCP将通过凝胶迁移率变动分析来鉴定;然后， 会被紫外线标记并净化。 一个核试验分析， 瞬时转染方法类似于用于鉴定 细胞因子转录物中富含AU的去稳定结构域将用于 确定mMCP转录物中调节细胞凋亡的特定核苷酸， 它们在mBMMC中的稳定性。 在具体目标2中，免疫化学和 生化技术将用于动力学实验来研究 某些细胞因子对翻译调节的影响， 蛋白酶在mBMMC中的颗粒积累瞬时转染和 将在大鼠中进行定点诱变实验 嗜碱性白血病细胞，以确定特定的氨基酸残基 在mMCP-5中，其控制其靶向颗粒及其相互作用 与丝甘肽蛋白聚糖。

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.