Exploring molecular targets for the establishment of drug therapy for the ossification of the posterior longitudinal ligament of the spine

为了验证P2Y1表达导致脊髓韧带骨化的假设，我们在OPLL和非OPLL患者的脊髓韧带细胞中强制表达P2Y1。实时定量聚合酶链反应和免疫荧光染色分别检测mRNA和蛋白的表达。转染P2Y1后，与非OPLL患者的细胞相比，来源于OPLL患者的脊髓韧带细胞中骨形态发生蛋白-2 （bone morphogenetic protein-2, BMP-2）和Sox9 mRNA的表达显著升高。免疫荧光分析显示，BMP-2和P2Y1仅在OPLL患者中表达升高，而Sox9在OPLL和非OPLL患者中表达升高。MRS2279是一种选择性P2Y1拮抗剂，可阻断P2Y1强制表达后Sox9和BMP-2的上调。此外，短暂转染P2Y1 4天后，仅在OPLL患者的脊髓韧带细胞中观察到矿化。这些结果表明P2Y1表达在OPLL患者脊柱韧带异位骨形成中起重要作用，调节P2Y1功能的药物将成为治疗OPLL的潜在候选药物。

To verify the hypothesis that P2Y1 expression causes ossification in the spinal ligaments, we forced expression of P2Y1 in spinal ligament cells obtained from OPLL and non-OPLL patients. The expression of mRNA and protein was investigated by quantitative real-time polymerase chain reaction and immunofluorescence staining, respectively. After transfection, bone morphogenetic protein-2 (BMP-2) and Sox9 mRNA expression was significantly increased in spinal ligament cells derived from OPLL patients compared with cells from non-OPLL patients 2 days after P2Y1 transient transfection. Immunofluorescence analysis showed that BMP-2 and P2Y1 expression was increased in OPLL patients only, while Sox9 expression was increased in OPLL and non-OPLL patients. MRS2279, a selective P2Y1 antagonist, blocked the upregulation of Sox9 and BMP-2 after forced expression of P2Y1. Furthermore, 4 days after transient transfection of P2Y1, mineralization was observed only in spinal ligament cells from OPLL patients. These results suggest that P2Y1 expression plays an important role in ectopic bone formation in the spinal ligaments of OPLL patients and that a drug which modulates the P2Y1 function will be a potential candidate for the drug in the therapy of OPLL.