FUNCTIONAL STUDIES OF OSTEOBLASTS

成骨细胞的功能研究

• 批准号: 2443674
• 负责人: Carol V Gay
• 金额: $ 27.12万
• 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 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2443674
• 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（改编自申请人的摘要）：长期目标 该研究计划的目的是了解体内骨细胞的功能。这 本申请中描述的研究目标是确定和 描述钙离子 (Ca++) 输送至部位的方式 的矿化作用。此前，首席研究员已表明 Ca-ATP酶大量存在于成骨细胞质膜中， 但其定向使得它不太可能将 Ca++ 分泌集中到 骨表面。因此，首席研究员将审查 将大量 Ca++ 驱出细胞，集中 Ca++ 的机制 进入矿化地点。覆盖骨表面的成骨细胞可以 被认为是一个连续的层，即间皮，它有能力 矢量易位离子。具体目标是：1）评估 比较培养的成骨细胞的极性发展和保留 含有新鲜分离的成骨细胞和完整的组织； 2）制定手段 可视化被认为聚集 Ca++ 的蛋白质的物理位置 使用免疫细胞化学进入矿化位点；- 3) 监测 使用新的方法从细胞（新鲜分离和培养）中流出 Ca++ 开发了荧光探针，可以捕获 Ca++，因为它从 细胞; 4）反向运行该过程以验证获得的结果 具体目标 3； 5) 监测分离血浆中 Ca++ 流出率 膜囊泡； 6) 确定地点和可能的监管 Na/K-ATPase（一种负责驱动 Ca++ 通量的酶）的机制 换取Na+。了解正常的机制 成骨细胞为细胞外骨提供钙提供基础 比较患病骨骼中的骨细胞功能。此外， 从这些研究中获得的新知识可能有助于最终的设计 对抗骨骼疾病的新疗法。