FUNCTIONAL STUDIES OF OSTEOBLASTS

描述（改编自申请人摘要）：长期目标 这项研究计划的主要目的是了解体内骨细胞的功能。的 本申请中描述的研究的目标是识别和 表征钙离子（Ca++）输送至部位的方式 的矿化。此前，主要研究者已经表明， Ca-ATP酶大量存在于成骨细胞质膜中， 但其取向使得其不太可能将Ca++分泌集中于 骨表面。因此，主要研究者将检查 将大量Ca++排出细胞的机制，将Ca++ 变成了矿化点覆盖骨表面的成骨细胞可以 被认为是一个连续的层，一个mesothelium，有能力， 使离子向量移位。具体目标是：1）评价 极性的发展，并保留在培养的成骨细胞相比， 用新鲜分离的成骨细胞和完整组织; 2）开发一种方法， 可视化蛋白质的物理位置， 进入矿化位点，使用免疫细胞化学;- 3）监测 Ca++流出细胞（新鲜分离和培养）使用新的 开发的荧光探针，可以捕获Ca++，因为它出现在 单元格; 4）反向运行该过程以验证在 具体目标3; 5）监测分离血浆中的Ca++流出率 膜囊泡;和6）确定位点和可能的调节 Na/K-ATP酶的机制，一种负责驱动Ca++通量的酶 交换Na+。理解正常的 成骨细胞为细胞外骨提供钙， 比较患病骨骼的骨细胞功能。此外该 从这些研究中获得的新知识可能有助于最终的设计 治疗骨骼疾病的新疗法。

DESCRIPTION (Adapted from applicant's Abstract): The long term objective of this research program is to understand bone cell function in vivo. The goal of the studies delineated in this application is to identify and characterize the means by which calcium ions (Ca++) are delivered to-sites of mineralization. Previously, the Principal Investigator has shown that Ca-ATPase is present in substantial amounts in osteoblast plasma membrane, but is oriented such that it is not likely to focus Ca++ secretion toward bone surfaces. Consequently, the Principal Investigator will examine mechanisms that drive Ca++ in large quantities out of cells, focusing Ca++ into sites of mineralization. Osteoblasts covering bone surfaces can be considered as a continuous layer, a mesothelium, that has the capacity to translocate ions vectorially. The specific aims are: 1) to evaluate polarity development, and retention in cultured osteoblasts as compared with freshly isolated osteoblasts and intact tissue; 2) to develop a means of visualizing the physical location of proteins believed to focus Ca++ into sites of mineralization using immunocytochemistry;- 3) to monitor Ca++ efflux from cells (freshly isolated and cultured) using a newly developed fluorescent probe that can trap Ca++ as it emerges from the cell; 4) to run the process in reverse to verify results obtained in Specific Aim 3; 5) to monitor rates of Ca++ efflux in isolated plasma membrane vesicles; and 6) to determine sites and possible regulatory mechanisms of Na/K-ATPase, an enzyme responsible for driving Ca++ fluxes in exchange for Na+. Understanding the mechanisms by which normal osteoblasts provide calcium to extracellular bone provides a basis for comparing bone cell function in the diseased skeleton. In addition, the new knowledge derived from these studies may assist in the eventual design of new therapeutics to combat skeletal diseases.