巨噬细胞M1/M2极化在椎间盘退变进程中的作用与机制研究

The regulation of immune homeostasis possesses potential significance for prevention and treatment of intervertebral disc degeneration (IDD) and its complications. Recently, macrophages were detected in IDD, but the biological role and regulation mechanism were unclear. It is proved that macrophage holding heterogeneity in cell phenotype (M1 or M2) and function. Based on that, we have previously constructed macrophage TSC1 knockout (TSC1KO) mice model (M1 polarization enhancement), and found that the M1 polarization of macrophages was significantly enhanced. By digital gene expression (DGE) profiling sequencing, we found Asporin expression was significantly up-regulated in TSC1KO macrophages. Aside from that, the clinical case specimens also found M1 polarization and Asporin expression were gradually increased in the IDD process, suggesting M1 polarization may promote IDD by secreting Asporin. Therefore, the aim of this study is to clarify the biological role of M1 or M2 polarization in the IDD process with TSC1KO mice (M1 polarization enhancement) and mTORC1 complex member RaptorKO mice (M2 polarization enhancement) in macrophages through animal IDD model and clinical IDD specimens. At the same time, the biological role and mechanism of M1 or M2 polarization in the process of IDD were further elucidated from the molecular level, cellular level and overall level by co-culturing macrophages with nucleus pulposus cells, as well as the injection of rhAsporin protein or its antibody within intervertebral disc. In conclusion, this study may shed light on the pathogenesis of IDD, and also provide potential targets for the prevention and treatment of IDD.

免疫稳态调控对椎间盘退变(IDD)及其并发症防治具有潜在的重要意义。研究发现IDD中存在巨噬细胞浸润，但作用与调控机制不清楚。巨噬细胞具有表型和功能异质性，课题组前期构建巨噬细胞敲除TSC1（TSC1KO）小鼠，发现其巨噬细胞发生M1极化，经DGE测序发现Asporin表达显著上调。前期病例标本检测也发现在IDD进程中M1极化与Asporin表达进行性增高，提示巨噬细胞M1极化可能通过分泌Asporin促进IDD。因此，本课题拟利用TSC1KO(M1极化)与RaptorKO(M2极化)小鼠与细胞，结合IDD动物模型与病例标本，明确巨噬细胞M1或M2极化在IDD进程中的作用。同时，通过巨噬细胞与髓核细胞共培养，以及椎间盘内注射rhAsporin蛋白或其抗体等方法，从分子、细胞与整体水平深入阐明巨噬细胞极化在IDD进程中的作用与细胞机制，以期为IDD发病机制提供新观点，为其防治提供潜在靶点。

研究发现IDD中存在巨噬细胞浸润，但作用与调控机制不清楚。巨噬细胞具有表型和功能异质性，课题组前期构建巨噬细胞敲除TSC1（TSC1KO）小鼠，发现其巨噬细胞发生M1极化。前期病例标本检测也发现在IDD进程中M1极化与Asporin表达进行性增高，提示巨噬细胞M1极化可能通过分泌Asporin促进IDD。因此，本课题拟利用TSC1KO(M1极化)与RaptorKO(M2极化)小鼠与细胞，结合IDD动物模型与病例标本，明确巨噬细胞M1或M2极化在IDD进程中的作用。同时，通过巨噬细胞与髓核细胞共培养，以及椎间盘内注射rhAsporin蛋白或其抗体等方法，从分子、细胞与整体水平深入阐明巨噬细胞极化在IDD进程中的作用与细胞机制，以期为IDD发病机制提供新观点，为其防治提供潜在靶点。.本课题通过3年努力，基本完成了预定的研究任务，并稍作修正。原课题提出巨噬细胞极化参与IDD的设想, 我们在研究中发现其不但与椎间盘退变程度、年龄增长等因素相关（在椎间盘退变过程中，M1细胞随着退变程度和年龄增加而减少，而M2细胞则增加，提示退变早期以促炎M1细胞为主，而退变晚期以抗炎M2型细胞为主），还和患者临床症状有显著相关性，因此我们扩大这部分工作，发现M1细胞标志CCR7+阳性率与术前腿痛VAS评分呈正相关，并在术后疗效不满意组中明显升高，提示术前椎间盘中，促炎M1巨噬细胞发挥了重要生物学作用。在转基因小鼠模型中，证实了我们的假设，即M1细胞活化的小鼠椎间盘退变呈现加速现象，而M2细胞活化小鼠椎间盘呈现延缓趋势。最后，我们将不同的巨噬细胞条件培养液，分别注入大鼠的尾椎间盘穿刺模型中发现M1CM可显著加速椎间盘退变，而M2CM则明显抑制椎间盘退变。髓核细胞体外共培养模型发现M1CM和M2CM均可促进炎性环境中髓核细胞增殖，但M2CM还可促进细胞外基质合成，抑制炎症因子产生，并且TGF-β1、IGF-1、GDF5表达升高，展现了其对IDD的修复潜力。本课题从细胞与整体水平阐明巨噬细胞极化在IDD进程中的作用及其细胞机制，为IDD发病机制提供新观点，为其个性化诊治提供新思路，为其再生修复提供新靶点。.