ASIC1a介导椎体终板软骨细胞凋亡小体在终板退变钙化中的作用机制研究

Formation of apoptotic bodies is associated with internucleosomal DNA fragmentation during cell apoptosis, which has been considered to play a key role in apoptosis-induced calcification. Some studies have demonstrated that chondrocyte derived apoptotic bodies may contribute to the pathologic vertebral cartilage endplate (CEP) calcification. In addtion, CEP calcification is one of the most important initial factors leading to intervertebral disc (IVD) degeneration. In the study of the last National Natural Science Foundation, We showed that ASIC1a, pH sensors in endplate chondrocytes, leads to apoptosis of endplate chondrocytes in IVD. Moreover, the activation of ASIC1a by acid can significantly promotes CEP apoptotic bodies and calcification in preliminary experiment. Therefore, we presume that ASIC1a can promote CEP endplate calcification through chondrocyte apoptotic bodies. Based on our previous research project, this research is in-depth （focusing on calcification from apoptosis) and innovation (discussion on new mechanism of ASIC1a-apoptosis body- CEP calcification). We will study the role of ASIC1a by ASIC1a selective agonist, antagonist, ASIC1a overexpression, ASIC1a RNA interference and protein microarray in the cell model,to analyze the effect of ASIC1a induced apoptotic body on endplate cartilage calcification and the signaling pathway; and the effect of blockage of ASIC1a on CEP and IVD degeneration in vivo by specific blocker of ASIC1a will be investigated. In general, the role of ASIC1a involved in the CEP calcification through apoptotic body may be clarified by these findings, based upon which the novel therapeutic strategies and theoretical basis for IVD degeneration could be provided.

凋亡小体是凋亡细胞的核DNA断裂为核小体片段,在凋亡诱导的钙化中起关键作用。软骨细胞凋亡小体可启动钙化的研究已有报道，而终板异常钙化可能是启动椎间盘退变的始动因素。在青年科学基金项目研究中，我们发现酸敏感离子通道1a（ASIC1a）是椎体软骨细胞膜的酸受体，介导酸诱导的终板软骨细胞凋亡。预实验示酸激活ASIC1a 能促进软骨细胞凋亡小体的形成和钙化发生。由此，我们推测：ASIC1a可通过凋亡小体来促进终板退变钙化作用。本研究是在上个项目基础上的深入（从凋亡聚焦到钙化）与创新（探讨ASIC1a-凋亡小体-终板钙化新机制），拟在细胞中通过ASIC1a 激动剂、拮抗剂、过表达、RNA 干扰及蛋白芯片等方法，分析ASIC1a介导凋亡小体在终板软骨钙化中的作用及信号通路，同时，通过动物模型进一步验证，意在探讨ASIC1a对终板软骨钙化的调控机制，为椎间盘终板退变钙化的防治提供新的理论基础和防治策略。

我们在前一个青年基金研究中，发现ASIC1a介导介导酸诱导的终板软骨[Ca2+]i水平的升高，ASIC1a通过H+-ASIC1a-Ca2+-下游信号转导通路介导软骨细胞凋亡。椎体终板软骨细胞凋亡在软骨终板钙化中起关键作用，实验结果进一步显示ASIC1a介导软骨细胞凋亡小体的发生，凋亡小体可以启动钙化的研究已有报道，而终板异常钙化引起椎间盘营养障碍，最终导致椎间盘退变。我们研究发现（1）酸敏感离子通道ASIC1a介导酸诱导的终板软骨细胞基质代谢损伤及机制；（2）酸敏感离子通道ASIC1a介导酸诱导的终板软骨细胞IκB-α磷酸化和p65亚基的移位；（3）ASIC1a通过NF-κB转录活性参与终板软骨细胞在细胞外酸性条件下的基质代谢；（4）发现ASIC1a介导椎体软骨细胞凋亡小体的发生，能够显著促进软骨细胞骨钙素的发生；（5）凋亡小体通过调控终板软骨细胞矿化基因ENPP1，ANK和TNAP的表达；（6）终板软骨细胞凋亡体通过焦磷酸(PPi)代谢途径增加钙化的作用机制。意在阐明酸敏感离子通道在终板软骨细胞的退变作用机制，对人们更好地认识酸敏感离子通道功能及寻找新的抗椎间盘退变的靶点有重要意义。