Effect of mechanical stress on osteogenesis facilitating by parathyroid hormone

甲状旁腺激素（PTH）是一种促进骨生成和骨吸收的激素。通过间歇性给予PTH刺激骨生成，并通过重复给药刺激骨吸收。然而，其机制尚不清楚。我们最近发现，流体切应力（机械应力之一）可诱导小鼠长骨骨细胞建立的MC 3 T3-Y 4-A2细胞中PTH受体mRNA的表达。为了阐明甲状旁腺素对成骨的影响，我们构建了甲状旁腺素受体与绿色荧光蛋白（GFP）的融合蛋白。首先，我们从MC 3 T3-Y 4-A2细胞的mRNA合成的cDNA中扩增了PTH受体的全长cDNA。然后，将PTH受体的全长cDNA连接到pEGFP-N1和pEGFP-C3载体（clontech公司）中。将携带PTH受体cDNA的EGFP载体转染小鼠成骨样细胞MC 3 T3-E1，用新霉素筛选稳定表达PTH受体/GFP融合蛋白的细胞。然而，没有分离出具有高荧光信号的细胞，因为具有明亮信号的细胞已经死亡。虽然原因尚不清楚，但它假设细胞中传递了过量的cAMP信号。最终分离出对新霉素具有抗性的细胞，但未观察到绿色荧光。RT-PCR检测融合蛋白mRNA的表达。我们正在用GFP检测机械应力对PTH受体表达的影响。

Parathyroid hormone (PTH) is a hormone that facilitate both osteogenesis and osteoclastic bone resorption. Osteogenesis was stimulated by intermittent administration of PTH, and bone resorption was atimulted repeated administration. However, its mechanism is unclear. We recently found that fluid shear stress, one of mechanical stress induced expression of mRNA of PTH receptor in MC3T3-Y4-A2 cells established from osteocytes in mouse long bone. In order to clarify the effects of PTH on osteogenesis, we constructed fusion protein with PTH receptor and Green fluorescence protein (GFP). First, we amplified a full length of cDNA of PTH receptor from cDNA that was synthesized from mRNA of MC3T3-Y4-A2 cells. Then, the full length of cDNA of PTH receptor was ligated into pEGFP-N1 and -C3 vectors (clontech co.). The EGFP vectors with PTH receptor cDNA were transfected into MC3T3-E1 oseoblast like cells established from mouse bone, and the cells stably expressing PTH receptor/GFP fusion protein were selected by neomysin. However, the cells that had high signals of fluorescence were not isolated because the cells that had bright signals were died. Though the reason was unclear, it assumed that excess signals of cAMP were transmitted in cells. The cells that had resistance to neomysin were finally isolated though the green fluorescence were not observed. The expression of mRNA of the fusion protein were identified by RT-PCR. We are examining the effects of mechanical stress on expression of PTH receptor with GFP.