髓核细胞老化及老化表型改变对局部微环境的旁分泌效应研究

Normal nucelus pulposus (NP) cells are essential for the maintance of homeostasis within intertebral disc(IVD). As NP cells gradually or prematurely become senescent and change to demonstrate senescent phenotypes, a significant loss of NP cells and their biological functions is expected to occur. In this study, replicative senescence and premature senescence are to be induced in human NP cells by serial subculturing and exposure to oxidative stresses respectively. The activation and crosstalk between the two senescent pathways, p53-p21-Rb and p16INK4A-Rb, will be detected and analyzed in senescent NP cells. By comparing the ability in producing inflammative cytokines, growth factors, matrix degrading enzymes, as well as the extracellular matrix between senescent and non-senescent NP cells, a comprehensive study of the senescent phenotypes of NP cells is made. Besides, normal NP cells will be co-cultured with senescent NP cells to observe the paracrine actions from the phenotype changes of senescent NP cells. Our studies aim to illuminate the role of NP cell senescence and the senescent phenotype during IVD degeneration. It is probable that aging and multiple stresses activate the p53-p21-pRB and/or p16INK4A-pRB senescent pathways to induce NP cell senescence, then the accumulation of senescent cells potentially deteriorates the disc microenvironment, which triggers and accelerates the IVD degeneration. By identifying the key element in the transduction of senescent pathways, our studies also support the feasiblity of anti-senescent approaches for IVD regeneration.

髓核细胞在维持椎间盘内环境稳定中发挥着核心作用，而细胞老化及老化后的表型改变可潜在导致髓核细胞数量减少和功能异常。本研究通过体外连续培养传代、氧化应激干预人正常髓核细胞，分别建立复制性老化和应激诱导的过早老化模型；探索P53-p21-Rb和p16INK4A-Rb通路在髓核细胞老化过程中的信号介导与交通；比较老化髓核细胞在炎症因子、生长因子、基质降解酶、细胞外基质合成四大方面的表型改变；观察老化髓核细胞旁分泌作用于正常髓核细胞后的致退变效应。从"细胞启动老化信号通路→形成复制性老化或过早老化→表达老化分泌表型→恶化邻近细胞生存微环境"角度认识髓核细胞老化及其表型改变在椎间盘退行性变中的病因学作用，寻找髓核细胞老化的关键调控信号，为生物学治疗更具针对性地选择抗老化靶点来有效修复椎间盘退变打开崭新思路。

髓核细胞老化在椎间盘退变过程中起着重要作用，而髓核细胞老化表型的改变及其对局部微环境的旁分泌效应尚不明确，明确椎间盘退变过程中髓核细胞老化表型的变化及其生物学作用将有助于深入认识椎间盘退行性变，为椎间盘退变的生物学治疗提供更多的干预靶点。本课题假说髓核细胞老化表型的改变通过对局部微环境的旁分泌作用影响椎间盘的退变与生物学修复。我们发现：.（1）髓核细胞老化过程中细胞表型发生显著改变，MMPs（MMP-1，-3，-10，-12及-13）、炎症因子（TNF-α、IL-1β、IL-6、IL-8）、bFGF表达上调，基质合成相关细胞因子（TGF-β1）表达下调。不同老化模型的表型改变具有一定的差异性，这种差异性表达提示不同退变或老化诱导因素下髓核细胞可形成特异性的分泌表型，而不同的表型可能与激活不同的老化信号途径相关。.（2）炎症因子TNF-α能够激活髓核细胞内NF-κB和MAPK信号通路促进髓核细胞增殖，提示在椎间盘退变的早期，低剂量的炎症因子可以促进髓核细胞增殖而过早发生复制性老化。.（3）椎间盘内源性干细胞可以向椎间盘内迁移并修复髓核细胞，该迁移过程潜在受到趋化信号轴介导（如HGF/c-met，SDF-1/CXCR4等）。因此可通过寻找并利用“迁移-修复”的相关信号轴促进椎间盘的干细胞治疗和修复。干细胞自身的老化现象也提示需要针对干细胞的抗老化能力来促进椎间盘退变的修复效率。.（4）髓核细胞老化表型的改变可通过旁分泌作用激活正常髓核细胞NF-κB和MAPK通路而促进细胞增殖，TGF-β1可减轻退变髓核组织分泌液对正常髓核细胞的致退变作用，提示细胞移植的同时结合细胞因子补充可能更好的修复椎间盘退变。.（5）坐骨神经痛患者血清促炎症细胞因子IL-6、IL-8、TNF-α以及抗炎症细胞因子IL-4、IL-10的表达水平与疼痛程度有一定相关性，提示髓核细胞老化表型的改变与临床症状潜在相关。.（6）细胞累积性复制所引起的复制性老化属于机体自然衰老的生理过程，但在椎间盘独特微环境因素作用下细胞老化速度被诸多内环境因素潜在加速,后者包括生长因子匮乏、氧化应激和炎症反应激活。急性椎间盘外伤、过度力学负荷、糖尿病、慢性吸烟等因素可以放大致老化效应，椎间盘的低血管化状态潜在制约老化细胞的免疫清除。老化细胞多以细胞丛性聚集，上调炎症因子并促进分解代谢，进一步恶化椎间盘微环境。