G-CSF调控软骨前体细胞内稳态的机制研究

The discovery of chondrogenic progenitor cells has provided a potential solution for articular cartilage regeneration, these stem cells harboring at superficial layer of cartilage are the source of fresh chondrocytes, which will replace senescent chondrocytes to maintain normal structure and function of cartilage. How to maintain these stem cells homeostasis, prevent exhaustion and make them differentiate continuously to generate fresh chondrocyte is the key step to success. We had previously isolated chondrogenic progenitor cells from articular cartilage, and repaired cartilage defect successfully by G-CSF (granulocyte colony-stimulating factor) induced chondrogenic progenitor cell homing,we also found that G-CSF can regulate homeostasis of chondrogenic progenitor cell through SIRT6 (sirtuin 6) depended pattern, but the mechanism is unclear. Base on literatures information and our previous research data, we speculated that G-CSF regulate chondrogenic progenitor cell homeostasis by adjusting expression of SIRT6 through MAPK/ERK signal pathway. To test this hypothesis, we plan to construct transgenic mouse for tracing the daughter cells of chondrogenic progenitor cell, combined disturbing different taches of this signal pathway both in vivo and in vitro experiments, to explore the detailed mechanism of signal pathway in this process. This study is expected to reveal the mechanism of G-CSF controlling chondrogenic progenitor cell homeostasis, enrich theoris about cartilage metabolism and initiation of osteoarthritis. This study also holds special hope for providing theoretical basis and therapeutic targets for treatment of osteoarthritis by activating autogeneous stem cells.

软骨前体细胞的发现使关节软骨再生成为可能，这些位于软骨浅层的干细胞是更新衰老软骨细胞的源泉，如何维护这些干细胞内稳态、防止耗竭并使其持续分化产生新鲜软骨细胞是骨关节炎治疗成功的关键。课题组前期从关节软骨内分离出软骨前体细胞，发现水凝胶携带G-CSF蛋白能够诱导前体细胞归巢并实现软骨缺损修复，进一步研究还发现G-CSF通过依赖SIRT6的方式调节软骨前体细胞内稳态，但具体机制不清楚。基于文献及前期工作基础，我们认为G-CSF很可能是通过MAPK/ERK信号通路调节SIRT6表达从而调控软骨前体细胞内稳态。为验证该假说，本项目拟构建转基因小鼠进行软骨前体细胞谱系追踪及细胞分选，联合体内外实验干扰上述信号通路不同位点，研究此过程中信号传导机制。该研究将揭示软骨前体细胞内稳态调控机制，丰富关节软骨代谢及骨关节炎发病机制等基础理论，为活化自体干细胞治疗早中期骨关节炎提供生物学理论基础和治疗靶点。

关节软骨是一种无血管、神经纤维及淋巴结构的低细胞密度组织，软骨细胞是关节软骨内的唯一细胞成分，通过合成细胞外基质维持关节软骨正常的解剖结构和生理功能，因此，软骨细胞衰老和更新直接决定着关节软骨的健康状态。然而，软骨细胞更新机制一直未能获得解答。本项目通过诱导细胞归巢、分离培养和鉴定证实成年哺乳动物关节软骨浅层有软骨前体细胞驻留，为原位诱导干/祖细胞归巢修复软骨损伤修复奠定了坚实的理论基础。同时，本项目研究证明G-CSF是软骨前体细胞维持内稳态的重要因素，G-CSF通过与细胞膜表面的G-CSF受体结合，激活细胞内MAPK/ERK信号通路导致c-Fos磷酸化激活进入细胞核内与Sirt6启动子结合促进其表达，从而调控下游靶基因表达以维持软骨前体细胞内稳态。此外，本项目研究发现条件性敲除csf3r基因将加重小鼠骨关节炎的严重程度，而关节腔注射小分子Sirt6激动剂MDL800能够逆转条件性敲除csf3r基因小鼠的骨关节炎进程，证明MDL800可能在骨关节炎治疗方面具有潜在的重要作用。本项目不但解决了关节软骨损伤修复时细胞来源的问题，阐明了软骨前体细胞调控维持内稳态的机制，而且发现小分子Sirt6激动剂MDL800具有显著的改善骨关节炎疾病进程的作用，为后续的基础和临床研究指出了方向，具有重要科学意义。