基于模式动物在体谱系示踪研究LepR+髓核细胞亚群及其在椎间盘退变中的作用

Intervertebral disc degeneration seriously endangers national health and living standards, but its pathogenesis remains to be clarified. The bottleneck of this puzzle is that the origin, heterogeneity，function and lineage fate of the nucleus pulposus cells (NPs) are not clear, and there are no genetic animal models with specificity especially on NPs sub-populations and therefore no related targeting biotherapeutics. The application is the first to discover that parts of NPs highly express Leptin receptor (LepR) through embryo-growth-mature periods. By establishing LepR-Cre;R26R;Tdtomato mice model for in vivo lineage tracing of the NPs, we are the first to identify LepR+ NPs sub-population, of which the percentage and quantity are markedly declined with aging. This project will first systematically clarify and distinguish the different temporal-spatial pattern and function of LepR+ NPs sub-population by using NPs-targeted genetic mice models; next, the fate mapping and dynamic function of LepR+ NPs will be checked and clarified in different mice models of disc degeneration and in human IDD samples. Furthermore, the essential role of LepR+ NPs under disc degeneration with its underlying mechanism will be figured out via using LepR-Cre;Progerinf/f mice. Finally, by using small molecule drugs, we will propose and validate the pathogenic LepR+ NPs-based early intervention strategies and prevention measures of disc degeneration.

椎间盘退变严重危害国民健康及生活水平，但其发病机制仍未阐明。瓶颈在于椎间盘髓核细胞的异质性、功能及命运转归尚不明确，并缺乏针对髓核细胞不同亚群标记的模式动物及靶向生物治疗药物。申请人首次发现部分髓核细胞在胚胎期（脊索细胞)-发育期-成熟期高表达瘦素受体（LepR），运用LepR荧光标记小鼠（LepR-Cre;R26R;Tdtomato）进行在体谱系示踪，首次鉴定出髓核细胞具有LepR+亚群，其数量随年龄增长与退变加重显著降低。本项目首先通过模式动物谱系示踪，系统观察LepR+亚群的时空间分布规律及功能状态；利用多种小鼠椎间盘退变模型与人椎间盘退变临床样本，阐明LepR+亚群在椎间盘退变中的命运转归及功能变化；运用模式动物特异性调控LepR+亚群凋亡/衰老，验证其在椎间盘退变中的关键作用；通过体内/外小分子药物干预，初步探索基于LepR+亚群靶向的椎间盘退变早期干预策略及疾病防治措施。

申请人结合前期研究基础，首次发现部分髓核细胞在胚胎期（脊索细胞）-发育期-成熟期高表达瘦素受体（LepR），通过构建LepR荧光标记小鼠（LepR-Cre;R26RtdTomato）进行在体谱系示踪，首次证实髓核细胞具有异质性，并鉴定出髓核细胞LepR+亚群。本项目首先通过体内功能鉴定及体外细胞分选验证，系统观察LepR+亚群从胚胎发育到成熟期的时空间分布、命运转归及功能状态；通过建立多种小鼠椎间盘退变模型（力学失稳、衰老退变LMNA G609G/G609G）与临床椎间盘退变患者不同分级髓核样本，进一步阐明LepR+亚群在椎间盘退变发生与发展中的病理性命运转归与功能变化；最后通过小分子药物干预提出并探索靶向潜在致病亚群的椎间盘退变早期干预策略及疾病防治措施。