Runx2/Nell-1反馈环路在骨关节炎钙化层重塑中的作用机制研究

Remodeling of calcified layer characterizes the early pathological change of osteoarthritis (OA). Nell-1 is a critical promoting factor of chondrocytes hypertrophy, but the detailed mechanism still remains unclear. Previous studies showed that Smurf2 might be involved in the Runx2 regulation by upper stream Nell-1. Based on this, we propose a hypothesis that Runx2/Nell-1 negative feedback loop regulates calcified layer remodeling. We plan to establish chondrocytes hypertrophy models combined with intervention methods to investigate the phosphorylation and degradation activity of Nell-1 on Smurf2. Ubiquitination modification is to be determined by the tandem mass spectrometry analysis. The transcription activity and stability of Runx2 will be determined by the luciferase report system and cycloheximide chasing methods. To further explore the influence of our hypothesized feedback loop on chondrocytes hypertrophy, IL-1 stimulated OA chondrocytes models will be adopted. In vivo studies will then be performed to confirm the effects of this feedback loop on the remodeling of calcified layer. This program tends to reveal the critical function of Nell-1 in the calcified layer construction from the sight of ubiquitination-proteosome aspect. Hopefully, we aim to provide more evidence on the theoretical and therapeutic significance of Nell-1 as a target in treating osteoarthritis.

钙化层重塑是骨关节炎（OA）早期的特征性病理改变，Nell-1为促钙化层软骨细胞肥大表型的关键因子，但其在重塑过程中详细机制不清。前期预实验显示，Smurf2可能参与Nell-1对上游基因Runx2的活性调节，继而影响细胞肥大化，故据此提出“钙化层软骨细胞Runx2/Nell-1反馈环路调控钙化层重塑”假说。拟利用软骨细胞肥大模型结合干预实验，观察Nell-1对Smurf 2磷酸化及蛋白降解的变化，利用串联质谱分析鉴定对Runx2泛素化修饰；并利用荧光素酶系统和CHX追逐法分别检测其对Runx2转录活性和蛋白稳定性的改变；进而利用白介素-1刺激模拟OA软骨细胞模型，探讨前述反馈环路对软骨细胞肥大化的影响；最后利用动物模型在体研究该环路对钙化层重塑的效应。本项目试从泛素-蛋白酶体途径揭示Nell-1对软骨细胞的作用，进一步揭示其在钙化层重塑的机制，以期为OA的防治提供重要的理论和实验依据。

钙化层重塑是骨关节炎（OA）早期的特征性病理改变，Nell-1为促钙化层软骨细胞肥大表型的关键因子，但其在重塑过程中详细机制不清。在本课题中，我们发现，Nell-1促进软骨细胞分化，同时也促进软骨细胞肥大化。Runx2可促进Nell-1表达，同时Nell-1可以经由Smurf2参与对Runx2的活性调节，该反馈环路决定软骨细胞的肥大分化，并影响钙化层的重塑过程，参与骨关节炎的发生发展。Nell-1对软骨细胞的作用，进一步揭示其在钙化层重塑的机制，实验结果将为OA的防治提供重要的理论和实验依据。