酸敏感离子通道3介导的细胞自噬在椎间盘炎症环境形成中的作用及机制研究

Abnormal expression of inflammation factors from nucleus pulposus cells raises inflammatory cells and leads to inflammatory environment of degenerated intervertebral disc. It is known that acid-sensing ion channels (ASICs) are sensors of cells to mornitor changes of extracellular hydrogen ions, and their activation lead to increased expression of inflammation factors. Also, several studies found that ASICs were involved in the activation of autophagy. We and other researchers found that autophagy participates in inflammation factors biosynthetic and secretory processes a variety of cells. Although one subtype of ASICs, ASIC3, is expressed in nucleus pulposus cells, whether ASIC3 contributes to the formation inflammatory environment in degenerated intervertebral disc is still unknown. Thus we raise a hypothesis: autophagy regulated by ASIC3 contributes to the formation of inflammatory environment in degenerated intervertebral disc. The present study attempts to, examine the influence of ASIC3 on inflammation factors biosynthesis and secretory in nucleus pulposus cells, explore the possible role of autophagy in ASIC3-mediated inflammation factors biosynthesis and secretory in nucleus pulposus cells, and analyze the mechanism in it, finally, observe the role of ASIC3 and autophagy inhibitors on inflammation factors expression and degeneration process of intervertebral disc. The purpose of the study is to clarify the role of autophagy regulated by ASIC3 on the formation inflammatory environment in degenerated intervertebral disc, which may promote the target to attenuate disc degeneration process.

髓核细胞异常表达炎症因子是募集炎症细胞、促进椎间盘炎症环境形成的始动因素。酸敏感性离子通道（ASICs）的激活引起多种细胞分泌炎症因子，也能促进细胞自噬水平增高。文献报道和课题组前期研究显示，应激引起的自噬在多种细胞中参与炎症因子表达。虽然髓核细胞表达ASIC3，但目前尚未证实椎间盘中是否存在以上现象。为此我们提出假说：酸性应激下，髓核细胞ASIC3激活，作用于细胞自噬过程，使自噬水平异常增高，引起髓核细胞表达炎症因子。本研究首先在模拟椎间盘酸性环境下，结合siRNA干扰技术和药物阻断作用，研究正常髓核细胞中ASIC3激活与炎症因子表达的相关性及ASIC3对自噬的激活作用；再通过对自噬水平的调控和下游信号通路的阻断，研究细胞自噬对炎症因子表达的调控及其分子机制；最后利用动物模型明确阻断ASIC3和抑制细胞自噬对炎症因子的表达和椎间盘退变的影响，旨在从炎症环境角度寻找新的靶点延缓椎间盘退变。

本研究首先利用退变髓核组织条件培养基干预正常髓核细胞，发现正常髓核细胞在退变环境中发生分泌表型改变，表达TNF-α、IL-1β和IL-6等炎症因子。接着将负载TNF-α拮抗剂rhsTNFRII的纳米粒通过化学键接枝于微流控多孔微球，原位构建拮抗剂功能化的可注射多孔微球，发现该抗炎微球能显著抑制退变髓核的炎症微环境，延缓椎间盘退变，并增强外源性种子细胞的治疗效率。基于以上研究基础，本研究通过对ASIC3和细胞自噬水平的调控，进一步深入探讨ASIC3和细胞自噬对炎症因子表达的调控及其分子机制，发现在椎间盘酸性微环境下，髓核细胞中ASIC3被激活，并作用于细胞自噬过程，使自噬水平异常增高，激活下游相关通路，促使髓核细胞分泌表型的改变，表达IL-1β、TNF-α和IL-6等前炎症因子。阻断ASIC3可抑制髓核细胞在酸性应激下表达炎症因子。最后，本研究通过微流控技术产生的GelMA微球共价修饰接枝ASIC3特异性阻断剂APETx2，并负载髓核细胞，设计了一种能够调节局部炎症反应的可注射“多肽-细胞-水凝胶”仿生微球。这种载细胞多肽微球不仅能够特异性长效阻断ASIC3，减少炎症因子表达，还能够提高负载髓核细胞的增殖活力，增强细胞外基质沉积作用，达到抑制椎间盘炎症反应的同时促进髓核组织再生。这为将来以ASIC3为靶点延缓椎间盘退变提供实验依据。总之，本研究以髓核细胞表达炎症因子为切入点，探索椎间盘炎症微环境的启动机制，并通过以ASIC3为治疗靶点，延缓或抑制炎症微环境的形成，延缓椎间盘退变，促进髓核再生。