MOLECULAR ASPECTS OF BONE FORMATION INDUCED BY MECHANICAL STRESS

取SD大鼠颅骨，解剖顶骨，冲洗软组织。在骨的顶部制作针孔，并将无菌夹子放入孔中以产生0.7g的向外力。分别在培养液中培养1、3、7 d和2周后，采用RT-PCR和原位杂交技术检测骨相关蛋白骨桥蛋白（OPN）、骨连接蛋白（ON）、骨钙素（OC）和骨形态发生蛋白4（BMP-4）mRNA的表达。在正常情况下，ON和OC由靠近矿化骨的成熟成骨细胞表达，ON也由靠近骨缝的增殖成骨细胞表达。ON和OC阳性细胞碱性磷酸酶也呈阳性。力负荷后，骨缝区的未成熟成骨细胞和矿化基质中的骨细胞表达OPN。成熟骨周围的静止成骨细胞中无OPN信号。RT-PCR检测骨形态发生蛋白-4（BMP-4）的表达，但在原位杂交中未检测到BMP-4的表达。

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.