REGULATION OF DIFFERENTIATION AND DEATH OF HUMAN AND MURINE OSTEOCLASTS

这份提案代表着一个已经成为一部分的项目的继续 我们的SCOR补助金自1987年开始以来，已经产生了21 到目前为止同行评审的论文。在第一个资助期，技术是 开发用于在动物模型(大鼠)中评估破骨细胞活性 和小鸡)。这种方法被用来检测破骨细胞的调节。 通过PTH、CT和pH测定活性。在第二个资助期， 认识到骨吸收的数量不仅取决于 单个破骨细胞的活性，但也取决于它们的募集率， 建立了评估破骨细胞形成的技术，以及 活性，在小鼠骨髓培养中。这种方法被用来研究 甲状旁腺素和雌激素的相互作用。虽然这些动物模型已经产生了 许多有用的数据，研究表明，有标记的物种 破骨细胞活性的差异，更重要的是，它们的 促钙因子的调节作用。这就强调了 如果我们有兴趣将结果外推到 正常和不正常的人类骨骼生物学。然而，直到最近， 人类破骨细胞的研究一直受到获取困难的阻碍 在培养中有足够数量的破骨细胞表达该键 破骨细胞的表型特征，即骨吸收。相当 最近，这个问题已经通过将M-CSF添加到 外周来源破骨细胞前体细胞的培养 血液循环和骨髓。在本项目中，我们建议 利用这些新的体外人类破骨细胞形成模型， 除了“基因敲除”的小鼠模型，增加我们对 破骨细胞分化和死亡的调控机制。这个 项目分为三个具体目标：1.调查 人破骨细胞分化的调控及分离纯化的A 融合前破骨细胞的数量。2.调查 雌激素受体α和β对小鼠和人的调节作用 破骨细胞形成。3.探讨细胞凋亡在细胞周期调控中的作用 雌激素对人和小鼠破骨细胞形成和寿命的影响。 尽管这个细胞生物学项目被设计为一个独立的项目，但它是 与SCOR的其他项目很好地结合起来，因为 两种重要的药物(甲状旁腺素和雌激素)将在#年进行研究。 体外培养破骨细胞。这个项目涉及的领域很多 与其他项目整合。例如，这个项目研究了 破骨细胞中骨桥蛋白合成的调节以及在项目2中 将在成骨细胞中进行研究。此外，将继续探讨的问题 在动物和临床研究中(例如，间歇给药 甲状旁腺素对破骨细胞募集的影响不同于连续 管理？)也将在这个项目中解决。

This proposal represents the continuation of a project that hs been a part of our SCOR grant since its inception in 1987 and which has resulted in 21 peer-reviewed papers to date. In the first funding period, techniques were developed to allow assessment of osteoclast activity in animal models (rat and chick). This approach was used to examine the regulation of osteoclast activity by PTH, CT, and pH. In the second funding period, with the recognition that the amount of bone resorbed depends not only on the activity of individual osteoclasts, but also on their rate of recruitment, techniques were established to assess osteoclast formation, as well as activity, in murine marrow cultures. This approach was used to study the interactions of PTH and estrogen. While these animal models have yielded much useful data, the studies have indicated that there are marked species differences in osteoclast activity and, more importantly, in their regulation by calciotropic factors. This emphasizes the importance of using human cells if were are interested in extrapolating the results to normal and abnormal human skeletal biology. Until recently, however, the study of human osteoclasts has been hampered by difficulties in obtaining sufficient numbers of osteoclasts in culture that express the key phenotypical feature of osteoclasts, namely bone resorption. Quite recently, this problem has been overcome by the addition of M-CSF to cultures of osteoclast precursor cells derived from the peripheral circulation and bone marrow. In this present project we propose to capitalize on these new in vitro models of human osteoclast formation, in addition to "knockout" murine models, to increase our understanding of the mechanisms regulating the differentiation and death of osteoclasts. The project is divided into three specific aims: 1. To investigate the regulation of human osteoclast differentiation and isolate a purified population of pre-fusion osteoclasts. 2. To investigate the role of estrogen receptors alpha and beta in regulating murine and human osteoclast formation. 3. To investigate the role of apoptosis in mediating estrogen's effects on human and murine osteoclast formation and life span. Although designed as an independent project, this cell biology project is well integrated with the other projects of the SCOR as the effects of the two agents of central interest (PTH and estrogen) will be studied in osteoclasts in vitro. There are numerous areas in which this project integrates with the other projects. For example, this project studies the regulation of osteopontin synthesis in osteoclasts and in Project 2 this will be studied in osteoblasts. Moreover, questions that will be pursued in the animal and clinical studies (e.g., does intermittent administration of PTH have different effects on osteoclast recruitment than continuous administration?) will also be tackled in this project.