EVALUATION OF BONE CELL FUNCTION BY CONFOCAL IMAGING

描述（来自申请书）

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.