NEW EXPERIMENTAL SYSTEM : CO-EXISTANCE OF BONE FORMATION AND RESORPTION IN VITRO

前列腺素E_2 （PGE_2）在骨中具有合成代谢作用，我们观察了PGE_2对培养大鼠骨髓基质细胞形成骨样矿化组织的影响。在培养早期添加PGE_2可增加矿化组织的数量，而在矿化前添加PGE_2可抑制矿化组织的形成，说明PGE_2的作用与成骨细胞分化的阶段有关。然后，我们试图找到PGE_2在该培养体系早期的合成代谢作用的信号转导途径，我们的实验揭示了camp依赖的信号转导参与了这一现象。另一方面，PGE_2增加细胞内Ca，并通过PGE受体亚型刺激或抑制cAMP的产生；EP_1, EP_2, EP_3。这些受体亚型最近被克隆出来。我们研究了这些受体亚型在骨组织中的表达。RT-PCR结果显示，EP_1、EP_2和EP_3在大鼠颅骨中表达，成骨细胞MC3T3-E1表达EP_1和EP_2。新生儿颅骨冷冻切片原位杂交分析发现，EP_2在成骨细胞和非骨接触细胞中表达，可能包括成骨前细胞。EP_2在颅骨发育早期及大鼠骨髓细胞培养中均有表达。PGE_2反应伴随cAMP的产生是成骨细胞的特征之一。我们的研究结果表明，这种表型出现在成骨细胞分化和骨发育的早期阶段。

Administration of prostaglandin E_2 (PGE_2) has anabolic effects in bone, we examined the effects of PGE_2 on bone-like mineralized tissue formation in rat bone marrow stromal cell culture. Addition of PGE_2 at early stage of culture increased the amount of mineralized tissue whereas addition of PGE_2 at later stage, just before mineralization, inhibited mineralized tissue formation, indicating that effects of PGE_2 is depending on the stage of osteoblastic differentiation. Then, we tried to find the signal transduction pathway of the anabolic effect of PGE_2 at the early stage of this culture system, and our experiment revealed the involvement of cAMP-dependent signal transduction in this phenomenon. On the other hand, PGE_2 increases intracellular Ca, and stimulates or inhibits cAMP production through PGE receptor subtypes ; EP_1, EP_2 and EP_3, respectively. These receptor subtypes have been recently cloned. We investigated the expression of these receptor subtypes in bone tissue. RT-PCR revealed that EP_1, EP_2 and EP_3 were expressed in rat calvaria and that osteoblastic cells (MC3T3-E1) expressed EP_1 and EP_2. In situ hybridization analysis using cryosection of neonatal calvariae revealed that EP_2 was expressed by osteoblasts and cells not in contact with bone, probably including preosteoblasts. EP_2 expression was observed at an early stage in calvarial development, and also in rat bone marrow cell culture. It has been established that PGE_2 response accompanying cAMP production is one of the characteristics of osteoblasts. Our results indicate that this phenotype appears at an early stage of osteoblastic differentiation and bone development.