DIFFERENTIATION OF MAST CELL PROGENITORS

肥大细胞体外分化伴随着大规模的 随着祖细胞变为IgE受体阳性， 颗粒状肥大细胞 许多表型变化发生在 肥大细胞和嗜碱性粒细胞特有的基因产物的表达。 尽管这些特征使研究成为一个有吸引力的模型系统 细胞分化，肥大细胞缺乏明确的祖细胞， 细胞（大多数研究者使用未分级的骨髓）和使用 IL-3产生长期肥大细胞培养物不允许 表型变化的窄聚焦窗口。 我们定义了一个 肥大细胞祖细胞分化的晚期定型阶段 就在制粒之前。 这种肥大细胞定向祖细胞不 需要IL-3的分化，但确实需要由 来自结缔组织如胚胎皮肤的成纤维细胞。 该模型 系统提供了独特的优势，以分析肥大细胞发生，一个过程， 在速发型超敏反应疾病的发展中很重要。 首先，体外分化系统将用于遵循 在晚期祖细胞中的离散变化， 因素 分离的肥大细胞定向祖细胞的制备将 用纯化的成纤维细胞衍生因子进行表型分析和触发。 将通过以下动员来监测细胞活化： 细胞内钙和氚化胸苷的掺入。 变化 在高亲和力IgE受体，生长因子受体， 组胺生物合成和颗粒组装将在顺序上相互关联 与信号转导有关。 第二，分化系统将适用于大鼠研究， 以检测糜酶、RMCP-II、羧肽酶A或肝素 胚胎皮肤上培养的肥大细胞中的蛋白多糖 单层。 这将使我们能够研究肥大细胞的异质性 （粘膜型与结缔组织型）。 第三，引起原始骨转变的因素 骨髓祖细胞的肥大细胞进展到IL-3的独立 定向祖细胞将通过旨在模拟 在体外，两种最可能的体内环境引起 定向祖细胞：来自肠道的完全脱颗粒的肥大细胞 粘膜引流到MLN，或骨髓祖细胞运输到 MLN作为原始祖先并发展成为承诺的 原位祖细胞。

Mast cell differentiation in vitro is accompanied by large-scale phenotypic changes as progenitors become IgE receptor-positive, granulated mast cells. Many of the phenotypic changes occur by expression of gene products unique to mast cells and basophils. Although these features make for an attractive model system for studies of cellular differentiation, the lack of a defined progenitor for mast cells (most investigators use unfractionated bone marrow) and the use of IL-3 to generate long term mast cell cultures has not allowed for a narrowly-focused window on the phenotypic changes. We have defined a late, committed stage in the differentiation of the mast cell progenitor just prior to granulation. This mast cell-committed progenitor does not require IL-3 of differentiation but does require factors provided by fibroblasts from connective tissue such as embryonic skin. This model system provides unique advantages to analyze mastocytogenesis, a process important in the development of diseases of immediate hypersensitivity. First, the in vitro differentiation system will be used to follow discrete changes in late-stage progenitor cells responding to defined factors. Preparations of isolated mast cell-committed progenitors will be phenotyped and triggered with purified fibroblast-derived factor. Cell activation will be monitored by following the mobilization of intracellular calcium and incorporation of tritiated thymidine. Changes in expression of high affinity IgE receptors, growth factor receptors, histamine biosynthesis, and granule assembly will correlate in sequence with signal transduction. Second, the differentiation system will be adapted for rat studies in order to detect chymase, RMCP-II, carboxypeptidase A, or heparin proteoglycan in mast cells derived from culture on embryonic skin monolayers. This will allow us to study mast cell heterogeneity (mucosal vs connective tissue type) in an in vitro system. Third, the factors which cause the transition of the primitive bone marrow progenitor for mast cells to progress to the IL-3 independent committed progenitor will be determined by experiments designed to mimic in vitro the two most likely in vivo milieus that give rise to the committed progenitor: completely degranulated mast cells from the gut mucosa draining into the MLN, or bone marrow progenitors trafficking to the MLN as a primitive progenitor and progressing into committed progenitors in situ.