BIOCHEMICAL CHARACTERIZATION OF OSTEOCLASTS

在过去几年中，积累了关于 破骨细胞的谱系和激素反应性，但很少 这些现象背后的生化机制 仍然知之甚少。 后一个信息差距是 特别是在分子生物学水平上， 存在关于分离、表征和/或 这些基因定义破骨细胞RNA的调控， 鉴定几个基因（例如，碳酸酐酶，其活性 相对于相关骨髓，破骨细胞出现升高， 骨髓来源的细胞，例如，巨噬细胞和巨噬细胞 多核巨细胞 在目前的更新中，三大 确定的目标，如果实现，应提供重要的 深入了解基因调控机制， 破骨细胞 一是进一步放大cDNA的“面板” 探索基因表达调控所必需的探针。 将采用两种战略来扩大小组成员： 一种是以前从非骨细胞或组织克隆的cDNA (e.g.,人抗酒石酸酸性磷酸酶）将用于 在鸡破骨细胞cDNA文库中鉴定其同源物; 另一种是cDNA克隆， 破骨细胞发育/功能相关基因将从 破骨细胞cDNA文库的比较和抗体筛选 使用cDNA探针和为基因开发的单克隆试剂 使用cDNA探针和单克隆试剂显著表达 为破骨细胞中显著表达的基因而开发， 相关细胞 第二个目的是确定基因的模式 表达式（即，特定mRNA丰度的变化） 分化破骨细胞和破骨细胞对选择性 效应物质 最后的目的是开始分析 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 使用核径流技术和转染 启动子-报告基因构建体进入破骨细胞和相关 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.