BIOCHEMICAL CHARACTERIZATION OF OSTEOCLASTS

在过去的几年里，关于 破骨细胞的谱系和激素反应性，但很少 例外，这些现象背后的生化机制 人们对此仍然知之甚少。后一种信息差距是 尤其是在分子生物学水平上，没有 文学存在于与世隔绝、刻画人物和/或 定义破骨细胞RNA和in的基因的调控 确定几个基因(例如，具有活性的碳酸酐酶 与相关骨髓相比，破骨细胞中的水平明显升高 骨髓来源的细胞，例如巨噬细胞和巨噬细胞 多核巨细胞。在这次更新中，主要有三个方面 确定的目标，如果实现，应该提供重要的 对基因调控机制的洞察 破骨细胞。一是进一步扩大cdna的“面板”。 探索基因表达调控所需的探针。 这种小组扩大将通过两项战略实现； 一种是先前从非破骨细胞或组织中克隆的cDNA (例如，人的抗酒石酸酸性磷酸酶)将用于 在鸡破骨细胞cDNA文库中鉴定它们的同源物； 另一种是对未知基因作出反应的基因克隆 破骨细胞发育/功能相关基因将从 破骨细胞文库的比较法和抗体筛选法 利用为基因开发的cdna探针和克隆试剂 使用cdna探针和单抗试剂显著表达 针对破骨细胞中显著表达的基因和 相关单元格。第二个目标是确定基因的模式 表达(即，特定的mRNA丰度的变化) 分化破骨细胞和对SELECT反应的破骨细胞 效应物。最终目标是启动对 基因表达变化的潜在机制 特别关注转录调控。这一目标将是 利用核径流技术和转基因技术实现的 启动子-报告基因构建进入破骨细胞及其相关 多核巨细胞。

Over the past several years, new data have accumulated on the lineage and hormonal responsivity of osteoclasts but, with few exceptions, the biochemical mechanisms underlying these phenomena remain poorly understood. The latter gap in information is particularly apparent at the molecular biological level where no literature exists on the isolation, characterization and/or regulation of those genes which define the osteoclast RNA and in identifying several genes (e.g., carbonic anhydrase whose activity appears elevated in osteoclasts relative to related marrow and marrow-derived cells, e.g., macrophages and macrophage multinucleated giant cells. In the present renewal, three major aims are identified that, if attained, should provide significant insight into the gene regulatory mechanisms operational in osteoclasts. The first is to further enlarge the "panel" of cDNA probes necessary to explore the regulation of gene expression. Such panel enlargement will be achieved using two strategies; in one, previously cloned cDNAs from non-osteoclastic cells or tissues (e.g., human tartrate resistant acid phosphatase) will be used to identify their homologs in chicken osteoclast cDNA libraries; in the other, cDNA clones responding to, as yet, unrecognized osteoclast development/function-related genes will be isolated from osteoclast cDNA libraries by comparative and antibody screening using cDNA probes and monoclonal reagents developed for genes prominently expressed using cDNA probes and monoclonal reagents developed for genes prominently expressed in osteoclasts and related cells. The second aim is to determine the pattern of gene expression (i.e., changes in specific mRNA abundance) in differentiating osteoclasts and in osteoclasts responding to select effector substances. The final aim is to initiate analysis of the mechanisms underlying the changes in gene expression with a particular focus on transcriptional regulation. This aim will be achieved using the nuclear runoff technique and the transfection of promoter-reporter gene constructs into osteoclasts and related multinucleated giant cells.