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

为了弄清后纵韧带骨化的原因，采用cDNA微阵列方法对培养的经单轴循环拉伸（机械应力）的人脊髓韧带细胞进行了全面分析。微阵列分析显示，通过循环拉伸，OPLL患者韧带细胞中碱性磷酸酶、骨钙素、I型胶原和Cbfa1等许多骨化标记基因上调，而正常韧带细胞中没有上调，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的敏感性也较高。这些结果表明，机械应力通过改变与骨代谢相关的各种基因的表达参与了OPLL的进展。P2受体亚型在OPLL细胞中的表达谱与在非OPLL细胞中的表达谱有较大差异。尤其是P2Y1在OPLL细胞中的表达量远高于非OPLL细胞，在SaOS2（骨肉瘤细胞系）中的表达量几乎相等。作为P2Y1激动剂的ATP增加了OPLL细胞中ALP和骨桥蛋白等成骨标志物的表达，而在非OPLL细胞中则没有。在p2y1特异性拮抗剂存在时，这些增加几乎减少。此外，机械应力显著增加了OPLL细胞的ATP释放。这些结果表明，ATP作为影响骨化过程的细胞外因子之一，通过P2Y1的病理生理作用是重要的。它们成为OPLL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.