EVALUATION OF BONE CELL FUNCTION BY CONFOCAL IMAGING

通过共焦成像评估骨细胞功能

• 批准号: 6055414
• 负责人: Carol V Gay
• 金额: $ 17.3万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6055414
• 关键词: aging; bone development; calcium flux; confocal scanning microscopy; fluorescent dye /probe; homeostasis; laboratory rat; osteoblasts; tissue /cell culture

DESCRIPTION (from Application) Although there is no animal model that completely duplicates human osteoporosis, aging rats have been found to develop osteopenia that is characterized by reductions in bone volume, mean bone density, and bone mineral and protein content. Consequently, in the studies proposed the aged rat will serve as the animal model. The goal of the studies described is to evaluate changes in intracellular calcium homeostasis during aging in bone forming cells, the osteoblasts. Osteoblasts will be isolated from 4 month (young adult) and 15 month (early old) rats. The cells will be obtained from three bony sites: the periosteum of long bone, trabecular bone of the metaphysis and vertebral trabecular bone in order to compare cells likely to show little change (periosteal) with those likely to show considerable change (trabecular). The isolated osteoblasts will be maintained as short-term primary cultures in order that the results will better reflect the in vivo state. The cells will be cultured in the presence and absence of 1,25(0H)2 vitamin D3 in order to determine if the changes found are reduced by this important skeletal regulatory agent. Using novel fluorescent probes, calcium ion fluxes and mitochondrial function will be evaluated by confocal microscope imaging techniques. Confocal microscopy is a powerful technology that allows localization and quantitation of metabolic events in living cells. One probe, calcium green-C18, which can be inserted into the plasma membrane, provides a means of detecting sites and rates of Ca++ efflux. Cytosolic Ca++ probes, namely fura-2 AM and calcium green-l AM, will be utilized to monitor changes in Ca++ release from intracellular stores. Another cytosolic Ca++ probe, calcium green-5N, which has a lower affinity for Ca++ will be used to detect changes in re-entry rates of Ca++ into intracellular stores. Finally, mitochondrial function will be assessed, by use of a membrane potential responsive cyanine dye, JC-1. Determining the level of mitochondrial activity is planned since the calcium pumping ATPases in the plasma membrane and intracellular stores require ATP. The studies have been designed to lay ground work for developing a new diagnostic tool that would help determine the extent of osteoblast dysfunction in cell outgrowths from biopsy material.

描述（来自应用程序） 尽管没有动物模型完全重复人类 骨质疏松症，衰老大鼠已发展为骨质减少症 其特征是骨体积减少，平均骨密度和骨骼的特征 矿物质和蛋白质含量。因此，在研究中提出了 老化的大鼠将用作动物模型。 研究的目标 所描述的是评估细胞内钙稳态的变化 在骨形成细胞的衰老过程中，成骨细胞。成骨细胞将是 从4个月（年轻）和15个月（早年）大鼠隔离。这 细胞将从三个骨位点获得：长长的骨膜 骨，小梁的小梁骨和椎骨小梁骨 为了比较细胞可能显示的细胞很少变化（骨膜） 那些可能显示出很大变化的人（小梁）。孤立 成骨细胞将以短期初级培养为准。 结果将更好地反映体内状态。细胞会 在存在和不存在1,25（0H）2维生素D3的情况下进行培养 确定所发现的变化是否通过这个重要的骨骼减少 监管代理。 使用新型的荧光探针，钙离子通量 线粒体功能将通过共聚焦显微镜评估 成像技术。共聚焦显微镜是一项强大的技术 允许在生活中定位和定量代谢事件 细胞。一个探针，钙绿-C18，可以插入 质膜，提供了一种检测CA ++的位点和速率的方法 外排。胞质Ca ++探针，即Fura-2 AM和钙绿-L AM， 将用于监视CA ++从细胞内释放的变化 商店。另一个胞质Ca ++探针，钙绿-5n，具有 较低的CA ++亲和力将用于检测重新输入率的变化 Ca ++进入细胞内存储。最后，线粒体功能将 可以通过使用膜潜在反应性氰胺染料来评估 JC-1。计划确定线粒体活动的水平，因为 质膜和细胞内的钙泵送ATPase 商店需要ATP。 研究旨在进行地面工作 用于开发一种新的诊断工具，以帮助确定 活检材料中细胞出生中成骨细胞功能障碍的程度。