Weinreb M, Shinar D, Rodan G A
Department of Bone Biology and Osteoporosis, Merck Sharp & Dohme Research Laboratories, West Point, PA.
J Bone Miner Res. 1990 Aug;5(8):831-42. doi: 10.1002/jbmr.5650050806.
Alkaline phosphatase (AP), osteopontin (OP), and osteocalcin (OC) are expressed during osteoblastic differentiation. However, previous studies suggested differences in the timing and possibly the site of their expression. In this study we used in situ hybridization to follow the distribution of these osteoblastic markers during bone development. Frozen sections of neonatal rat long bones and calvariae were hybridized with 35S-labeled RNA probes complementary to the AP, OP, and OC mRNAs. Controls included sections hybridized with the sense (mRNA) probes or pretreated with RNase. Positive cells were identified in all areas of bone formation of the long bones and calvariae. Based on quantitative silver grain distribution and density, high levels of OP expression were present only in osteoblasts in close proximity to bone (one to two cell layers). OC expression, apparently at lower levels than OP, was also localized to osteoblasts in contact with bone. In contrast AP, which was expressed at lower levels than OP, was present in a large number of cells, including preosteoblasts that were many layers removed from the bone-forming surface. These findings are consistent with the asynchronous expression of phenotypically related genes and suggest that AP is an earlier differentiation marker than OP and OC during the formation of endochondral and membranous bone.
碱性磷酸酶（AP）、骨桥蛋白（OP）和骨钙素（OC）在成骨细胞分化过程中表达。然而，先前的研究表明它们在表达时间以及可能的表达部位存在差异。在本研究中，我们使用原位杂交技术来追踪这些成骨细胞标志物在骨骼发育过程中的分布。将新生大鼠长骨和颅骨的冰冻切片与与AP、OP和OC mRNA互补的35S标记RNA探针杂交。对照包括与正义（mRNA）探针杂交或用核糖核酸酶预处理的切片。在长骨和颅骨的所有骨形成区域均鉴定出阳性细胞。根据定量银颗粒分布和密度，高水平的OP表达仅存在于紧邻骨的成骨细胞中（一到两层细胞）。OC表达水平明显低于OP，也定位于与骨接触的成骨细胞中。相比之下，AP表达水平低于OP，存在于大量细胞中，包括从骨形成表面移除多层的前成骨细胞。这些发现与表型相关基因的异步表达一致，并表明在软骨内骨和膜性骨形成过程中，AP是比OP和OC更早的分化标志物。
