FUNCTIONAL STUDIES OF OSTEOBLASTS

成骨细胞的功能研究

• 批准号: 6176825
• 负责人: Carol V Gay
• 金额: $ 29.89万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6176825
• 关键词: 1,25 dihydroxycholecalciferol; antibody; calcium; calcium flux; calcium transporting ATPase; cell membrane; cellular polarity; chickens; confocal scanning microscopy; dexamethasone; fluorescent dye /probe; glucocorticoids; immunocytochemistry; membrane transport proteins; normal ossification; osteoblasts; osteocytes; protein sequence; sodium; sodium potassium exchanging ATPase; tissue /cell culture; vesicle /vacuole

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.

描述（根据申请人的摘要改编）：长期目标 该研究计划的体内是了解骨细胞功能。这 在本应用程序中描述的研究的目标是识别和 表征钙离子（Ca ++）的均值 矿化。以前，首席调查员表明 CA-ATPase在成骨细胞质膜中以大量存在 但要定向，因此不太可能将CA ++分泌集中在 骨表面。因此，首席调查员将检查 将CA ++大量驱动到细胞的机制，聚焦于Ca ++ 进入矿化部位。覆盖骨表面的成骨细胞可以是 被认为是一个连续层，一个具有能力的间皮 载体载体载体。具体目的是：1）评估 极性发展，并保留在培养的成骨细胞中 新鲜分离的成骨细胞和完整的组织； 2）开发一种手段 可视化蛋白质的物理位置，被认为聚焦Ca ++ 使用免疫细胞化学的矿化部位； -3）监测 使用新的 开发的荧光探针可以捕获Ca ++，因为它从 细胞; 4）反向运行该过程以验证在 具体目标3; 5）监测分离的等离子体中Ca ++外排的速率 膜囊泡； 6）确定位点和可能的调节 Na/k-ATPase的机制，一种负责驱动Ca ++通量的酶 换取Na+。了解正常的机制 成骨细胞为细胞外骨提供钙提供了基础 比较患病骨骼中的骨细胞功能。另外， 这些研究得出的新知识可能有助于最终设计 与骨骼疾病作斗争的新疗法。