PGP激活CXCR1/2促进软骨终板干细胞向髓核内归巢并诱导其向促炎症表型转化的机制研究

Our research group has been focusing on the Carilage Endplate derived Stem Cells (CESCs) related research, and we found that CESCs had robust potential to regenerate the degenerated nucleus pulposus (NP) when serving as seed cells for NP tissue engineering. However, It is still unknown what causes CESCs homing to NP and what is the subsequent biological effect of CESCs after homing. In our previous study, we found a positive correlation between the degree of intervertebral disc (IVD) degeneration and proline-glycine-proline(PGP) content in NP, which is a tripeptide produced by collagen degradation. Besides, CESCs expressed CXCR1/2, receptors for PGP, and PGP could induce CESCs migration. Finally, PGP could improve the ability of CESCs to degrade collagen and upregulate the expression of some important inflammatory factors significantly. So we raise the hypothesis that PGP could recruit CESCs homing to NP and make CESCs transformed into inflammatory phenotype by reacting with CXCR1/2. In this research, we will use transwell method, rabbbit IVD puncture model, et al to confirm the above-mentioned hypothesis. These findings will reveal a possible IVD degeneration mechanism and pave the way for exploring the biological therapy approach for IVD degeneration.

本课题组在人椎间盘软骨终板干细胞（CESCs）序列研究的基础上发现：CESCs具有较强的修复退变髓核组织的能力。然而，作为椎间盘内源性干细胞，CESCs向髓核归巢的调节机制，以及归巢后是发挥其修复潜能还是产生“促退变”效应仍不清楚。前期工作发现，人退变髓核组织中三肽PGP含量与退变程度具有相关性，以及CESCs表面表达PGP的受体CXCR1/2，且PGP可能通过PI3K/Akt－NF-κB信号通路对CESCs产生趋化作用。此外，PGP刺激CESCs 能够促进其对胶原蛋白的降解能力以及上调炎症因子的表达。于是猜测PGP通过CXCR1/2促使CESCs向髓核内归巢并诱导其向促炎症表型转化而发挥“促退变”效应。本课题拟通过迁移实验、兔椎间盘注射模型等方法进一步证实上述猜想，为椎间盘内源性干细胞的归巢机制及其在椎间盘退变中发挥的生物学效应提供理论基础，为椎间盘退变的生物学治疗途径提供新的思路。

椎间盘髓核细胞的衰老与IDD的发生和发展密切相关，我们之前的研究发现N-Ac-PGP参与了IDD的过程，但是它在IDD中的作用还没有得到很好的了解。为了进一步了解N-Ac-PGP在IDD中的具体作用机制，我们使用大鼠髓核(NP)细胞，探究N-Ac-PGP通过结合CXCR1诱导NP细胞过早衰老。本研究发现N-Ac-PGP可诱导NP细胞早衰，增强其基质分解代谢和炎症级联。此外，衰老的NP细胞经皮内传递至大鼠尾椎椎间盘加快了IDD的进程，N-Ac-PGP增大了衰老NP细胞对IVD内环境稳定的负向影响。我们发现：1、N-Ac-PGP通过CXCR1诱导NP细胞过早衰老；2、N-Ac-PGP增强了NP细胞的DNA损伤，并通过CXCR1激活了p53-p21-Rb通路；3、N-Ac-PGP通过CXCR1促进ROS生成，上调NP细胞中p16的表达；4、复制性衰老模型中与SASP相关的蛋白在mRNA水平上受到调控；5、N-Ac-PGP增强了NP细胞的分解代谢和炎症级联；6、N-Ac-PGP增强了衰老NP细胞对IVDs的破坏作用。我们的研究表明，N-Ac-PGP通过诱导椎间盘细胞过早衰老和激活椎间盘细胞的分解代谢和炎症级联，在IDD的发病机制中起关键作用。N-Ac-PGP也会破坏椎间盘细胞的氧化还原环境。因此，N-Ac-PGP是IDD新的潜在治疗靶点