FUNCTIONAL STUDIES OF OSTEOBLASTS

成骨细胞的功能研究

• 批准号: 2897012
• 负责人: Carol V Gay
• 金额: $ 28.74万
• 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/2897012
• 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(改编自申请者摘要)：长期目标 这项研究计划的目的是了解骨细胞在体内的功能。这个 本申请书中描述的研究的目标是确定和 描述钙离子(Ca++)被输送到-部位的方式 矿化的可能性。此前，首席调查员已经表明， CA-ATPase大量存在于成骨细胞质膜中， 但它的定位是不太可能将钙分泌集中到 骨骼表面。因此，首席调查员将审查 将大量钙离子赶出细胞，聚焦于钙离子的机制 变成矿化的地点。覆盖在骨表面的成骨细胞可以 被认为是连续的一层，一层间皮，具有 在垂直方向上转移离子。具体目标是：1)评价 培养成骨细胞的极性发育和滞留的比较 新分离的成骨细胞和完整的组织；2)开发一种方法 可视化被认为集中在Ca++上的蛋白质的物理位置 利用免疫细胞化学检测矿化部位；-3)监测 细胞(新鲜分离和培养)钙离子外流使用新的 开发了一种荧光探针，可以在Ca++从细胞内出现时捕获它 单元格；4)反向运行该过程以验证在 特定目标3；5)监测离体血中钙离子流出速率 膜泡；以及6)确定位置和可能的调节 钙离子外流驱动酶Na/K-ATPase的作用机制 以换取Na+。了解正常运行的机制 成骨细胞为细胞外骨骼提供钙提供基础 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。