MOLECULAR ASPECTS OF BONE FORMATION INDUCED BY MECHANICAL STRESS

取SD大鼠颅骨，解剖顶骨，冲洗软组织。在骨头顶端打上销孔，并在孔内放置消毒的夹子，以产生0.7g的外力。分别在α-MEM中孵育1、3、7d和2周，用RT-PCR和原位杂交的方法检测骨桥蛋白(OPN)、骨连接蛋白(ON)、骨钙素(OC)和骨形态发生蛋白4(BMP-4)等骨相关蛋白的表达。在正常情况下，ON和OC由矿化骨附近的成熟成骨细胞表达，ON也由向骨缝方向增殖的成骨细胞表达。On和OC阳性细胞也呈碱性磷酸酶阳性。在LAOD作用下，骨桥蛋白主要由增殖缝合区的未成熟成骨细胞和矿化基质中的骨细胞表达。成熟骨周围静息的成骨细胞内未见OPN信号。加载后即刻和矿化早期，RT-PCR检测到BMP-4的表达，但在ISH中没有发现组织学信号。进一步的研究正在进行中，包括对OPN的ISH方法的改进。

Calvaria of SD rats were removed out and parietal bones were dissected and washed of soft tissue. Pin holes were made at the top of bones and sterilized clips were set into the holes to make 0.7g of outerward force. Those bones were incubated in alphaMEM for 1,3,7 days and upto 2 weeks and expression of mRNA of bone related proteins, such as Osteopontin (OPN), Osteonectin (ON), Osteocalcin (OC) and Bone Morphogenetic Protein 4 (BMP-4), were investigated by the mathods of RT-PCR and in situ hybridization (ISH). On normal condition, ON and OC were expressed by mature osteoblasts a djacent to minerized bone and ON were also expressed by proliferating osteoblasts toward the suture. Those ON and OC positive cells were also positive for Alkaline phosphatase. After the force was laod, OPN were expressed by immature osteoblasts in proliferating suture zone and by osteocytes embedded in mineralized matrix. There was no OPN signal in resting osteoblasts around the mature bone. BMP-4 were detected by RT-PCR just after the load and during the early phase of mineralization, however, no histological signal was found in ISH.Further study is under investigation including the modification of ISH methodlogy for OPN.