Analysis of genes related to the ectopic bone formation induced by mechanical stress and search for drugs targeting the genes.

为了阐明后纵韧带骨化的原因，利用基因芯片技术对单轴循环拉伸(机械应力)培养的人脊柱韧带细胞进行了全面的分析。基因芯片分析显示，在OPLL患者的韧带细胞中，许多成骨标志物基因，包括碱性磷酸酶、骨钙素、I型胶原和Cbfa1，在周期性拉伸中上调，但在正常韧带细胞中不表达，这表明OPLL细胞对机械应力的敏感性高于正常韧带细胞。OPLL患者的韧带组织对机械应力也有较高的敏感性。这些结果提示，机械应激通过改变骨代谢相关基因的表达参与OPLL的进展。OPLL细胞和非OPLL细胞中P2受体亚型的表达谱有很大不同。尤其在OPLL细胞中，P2Y1的表达明显高于非OPLL细胞，而在SaOS2(骨肉瘤细胞系)中的表达几乎与之相当。作为P2Y1激动剂的三磷酸腺苷增加了成骨标记物ALP和骨桥蛋白在OPLL细胞中的表达，但在非OPLL细胞中不表达。这些增加在P2Y1特异性拮抗剂的存在下几乎被减弱。此外，机械应力显著增加OPLL细胞的ATP释放量。这些结果表明，作为影响骨化过程的细胞外因素之一，ATP通过P2Y1发挥重要的病理生理作用。它们成为OPLL的一个新的药物治疗靶点。

To clarify the cause of the ossification of posterior longitudinal ligament (OPLL), comprehensive analyses by cDNA microarray method were carried out using cultured human spinal ligament cells that had been subjected to uniaxial cyclic stretching (mechanical stress). Microarray analyses showed the up-regulation of many ossification-marker genes including alkaline phosphatase, osteocalcin, collagen type I and Cbfa1, by cyclic stretching in ligament cells from OPLL patients but not in normal ligament cells, suggesting that OPLL cells are higher sensitive to mechanical stress than normal ligament cells. The higher sensitivity to mechanical stress was also confirmed in ligament tissue from OPLL patients. These results suggest that mechanical stress participates in the progression of OPLL through changes in expressions of various genes related to bone metabolism.Spectrum of P2 receptor subtype expression in OPLL cells was quite different from that in non-OPLL cells. Especially, P2Y1 expression in OPLL cells was much higher than that in non-OPLL cells and almost equal to that in SaOS2 (osteosarcoma cell line). ATP as a P2Y1 agonist increased the expressions of osteogenic markers such as ALP and osteopontin in OPLL cells but not in non-OPLL cells. These increases were almost diminished in the presence of P2Y1-specific antagonist. Furthermore, mechanical stress increased ATP release from OPLL cells remarkably. These results suggest that as one of extracellular factors influencing the process of ossification, a pathophysiological action of ATP via P2Y1 is important. They become a novel pharmacotherapeutic target for OPLL.