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（来自申请） 虽然目前还没有完全复制人类的动物模型 骨质疏松症，已发现老龄大鼠出现骨质减少，即 其特征是骨量、平均骨密度和骨量减少 矿物质和蛋白质含量。因此，在研究中提出 老年大鼠将作为动物模型。 研究目标 描述的是评估细胞内钙稳态的变化 在骨形成细胞（成骨细胞）的衰老过程中。成骨细胞将会 从 4 个月（年轻的成年）和 15 个月（早期）的大鼠中分离出来。这 细胞将从三个骨部位获得：长骨的骨膜 骨、干骺端骨小梁和椎体骨小梁 为了比较可能表现出微小变化的细胞（骨膜）与 那些可能表现出相当大变化的人（小梁）。孤立的 成骨细胞将作为短期原代培养物进行维持，以便 使结果能够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，以便 以确定发现的变化是否因这一重要的骨骼而减少 监管代理。 使用新型荧光探针、钙离子通量 并通过共聚焦显微镜评估线粒体功能 成像技术。共焦显微镜是一项强大的技术 允许对活体代谢事件进行定位和定量 细胞。一个探针，钙绿-C18，可以插入 质膜，提供了一种检测 Ca++ 位点和速率的方法 外流。胞质Ca++探针，即fura-2 AM和钙绿-l AM， 将用于监测细胞内 Ca++ 释放的变化 商店。另一种胞质 Ca++ 探针，钙绿-5N，具有 对 Ca++ 的较低亲和力将用于检测重新进入率的变化 Ca++ 进入细胞内储存。最后，线粒体功能将 通过使用膜电位响应花青染料进行评估， JC-1。计划测定线粒体活性水平 质膜和细胞内的钙泵ATP酶 商店需要 ATP。 这些研究旨在奠定基础 开发一种新的诊断工具，有助于确定 活检材料中细胞生长物中成骨细胞功能障碍的程度。