Ihh信号通路在周期性机械应力刺激诱导黄韧带骨化中的作用机制

Ossification of ligamentum flavum (OLF) is one of the main causes of thoracic myelopathy. It is reported OLF is closely correlated with the increased local stress caused by the instability of the spine; however, the exact mechanism is unclear. Indian hedgehog (Ihh) signaling pathway is one of the important signaling pathways in bone development, which regulate cell differentiation by development of the concentration gradient. We have found significant overexpression of Ihh in OLF samples. Furthermore, application of cyclic mechanical stress to OLF cells resulted in significant increases in mRNA expression levels of Ihh and the ossification markers, while inhibition of Ihh resulted in the inhibited increase in mRNA expression levels of the ossification markers. Thus, we concluded that Ihh play an important role in OLF development. In this study, we will firstly examine the secretion regulatory elements of Ihh, and the expression of proteins of the Ihh pathway in OLF sample. Then, silence the genes of the secretion regulatory elements of Ihh and the genes of key proteins of the Ihh pathway, to confirm the mechanisms of development of the concentration gradient and the signal transduction of Ihh subjected to cyclic mechanical stress. Finally, establish an animal model of OLF induced by cyclic mechanical stress by using Ihh gene silence mouse, to test the above mechanisms. This study could provide a new clue to pathogenesis and treatment of OLF.

黄韧带骨化是一种能引起脊髓严重受压的致瘫性疾病，其发生和进展与脊柱失稳引起的局部应力增加密切相关，但具体分子机制不清。Ihh信号通路是骨生长发育的重要信号通路之一，通过浓度梯度来调控细胞行为。我们前期实验发现黄韧带骨化标本中Ihh的表达异常增高。对骨化的黄韧带细胞进行周期性机械应力刺激后，Ihh和骨化标记物的mRNA同时明显上调。抑制Ihh信号通路后，骨化标记物mRNA的上调受到抑制。我们推测：Ihh信号通路参与了周期性机械应力刺激诱导黄韧带骨化的过程。本研究拟在黄韧带骨化的病理组织水平检测Ihh分泌调控分子及Ihh信号通路相关蛋白的表达。在细胞水平分别沉默Ihh分泌调控分子及信号通路中关键蛋白的基因，明确周期性应力刺激诱导黄韧带骨化过程中Ihh的浓度梯度形成机制和信号传导机制。通过构建Ihh基因沉默小鼠的黄韧带骨化模型进一步验证上述机制。本研究将为黄韧带骨化的发病机制及治疗提供新的思路。

黄韧带骨化是一种能引起脊髓严重受压的致瘫性疾病，其发生和进展与脊柱失稳引起的局部应力增加密切相关，但具体分子机制不清。Ihh信号通路是骨生长发育的重要信号通路之一。通过本课题研究，主要有以下三方面的发现:一、胸椎黄韧带骨化患者病理标本中，骨化黄韧带组织中有骨小梁及新生血管形成，Ihh信号通路关键蛋白Disp、Ihh、Smo及Gli1为阳性表达。二、细胞层面研究发现，骨化的黄韧带细胞中Ihh信号通路关键蛋白Ihh、Smo、Gli1及骨化标记物Runx2, Osterix, ALP和OCN表达要显著高于正常的黄韧带细胞；对细胞加载15%的应力刺激后，其Ihh的促分泌蛋白Disp，Ihh信号通路关键蛋白及骨化标记物均显著增高；同时Ihh抑制剂能显著降低此变化。三、建立应力刺激胸椎黄韧带的大鼠模型，发现周期性应力刺激能引起胸椎黄韧带细胞Ihh及部分骨化标记上调，但在组织病理中并未见到明显的骨小梁形成。该课题证实应力刺激能引起黄韧带细胞的Ihh的分泌增加，进而导致骨化的形成。该研究有助于研发阻止或延缓骨化进展的药物，具有临床实用价值及社会经济效益。