SMSCs源外泌体负载吡格列酮通过PPARγ促骨关节炎软骨稳态平衡的机制研究

The pathological basis of osteoarthritis (OA) is the deregulation of articular cartilage, and the pathogenesis is unknown and there is no effective treatment in clinic. Synovial mesenchymal stem cells (SMSCs) can promote the regeneration of chondrocytes, while the main function is mediated by exosome and the secretion of extracellular matrix is insufficient. The preliminary studies have shown that PPARγ could not only inhibits autophagy of chondrocytes, but also promotes the expression of extracellular matrix, which plays an important role in the regulation of articular cartilage; mTOR mediated down-regulation of ULK1 and BECN1 expression in chondrocyte is closely related to OA. Can the exosomes of SMSCs loaded with pioglitazone (PPARγ activator) promote functional cartilage formation? What is the relevant mechanism? Preliminary experiments have confirmed that the exosomes of SMSCs loaded with pioglitazone can inhibit the expression of mTOR in chondrocyte. Hypothesis is put forward based on literature: the exosome of SMSCs loaded with pioglitazone can up-regulate PPARγ through the mTOR-ULK1-BECN1 functional center and secret extracellular matrix through mTOR-independent pathway to promote cartilage homeostasis. In this study, both the granular cartilage culture system and animal model are used to demonstrate the mechanism of promoting functional chondrogenesis, which will lay a foundation for the precise treatment of OA.

骨关节炎（OA）以关节软骨稳态失衡为病理基础，其发病机制不详，无有效的治疗方法。滑膜间充质干细胞（SMSCs）可促进OA软骨细胞再生，主要是通过外泌体实现，但存在细胞外基质生成不足的问题。我们前期研究发现PPARγ可增强软骨细胞自噬，促进细胞外基质分泌；mTOR介导软骨细胞ULK1和BECN1表达下调与OA密切相关。能否通过SMSCs源外泌体负载吡格列酮（PPARγ激活剂）促进功能软骨生成？作用机制是什么？预实验证实负载吡格列酮的SMSCs源外泌体可抑制软骨细胞mTOR表达。因此提出假设：负载吡格列酮的SMSCs源外泌体可上调PPARγ，调控mTOR-ULK1-BECN1功能中心，并通过mTOR非依赖性途径促进细胞外基质生成，维持软骨稳态平衡。本研究将应用手术诱导不稳定内侧半月板(DMM)OA模型和OA-iPSC颗粒软骨培养体系阐明其维持软骨稳态平衡的机理，为OA精准治疗奠定基础。

骨关节炎（OA）以关节软骨稳态失衡为病理基础，其发病机制不详，尚无有效的治疗方法。一方面，目前临床上用于治疗OA的方法无法阻止OA的组织学进展，另一方面，软骨的自我修复能力差、无法对大于5 mm的局部缺损进行修复，而目前的治疗方法均不能使受损软骨愈合形成自然状态的软骨组织。这就迫切需要我们寻求实现功能软骨生成的办法。滑膜间充质干细胞（SMSCs）可促进OA软骨细胞再生，主要是通过外泌体实现，但存在细胞外基质生成不足的问题。我们前期研究发现PPARγ可增强软骨细胞自噬，促进细胞外基质分泌；mTOR介导软骨细胞ULK1 和BECN1表达下调与OA密切相关。故本课题预想负载吡格列酮（PPARγ激活剂）的SMSCs源外泌体可上调PPARγ，调控mTOR-ULK1-BECN1功能中心促进细胞外基质生成，以实现维持软骨稳态平衡、促进功能软骨生成。本研究应用手术诱导不稳定内侧半月板(DMM) OA模型和OA-iPSC颗粒软骨培养体系进行体内、外研究，将负载吡格列酮的SMSCs源外泌体分别注入DMM OA模型鼠的关节腔，并将其与OA-iPSC颗粒软骨共培养，取OA膝关节进行micro-CT检测，取软骨组织应用Western blot及PCR进行自噬、凋亡、炎症及代谢相关分子检测。研究证实，负载吡格列酮的SMSC EVs可以上调PPAR-γ和LC3II/LC3I的表达，抑制mTOR的表达，上调p62、Col2a1和下调MMP13表达，进而实现调节基质相关基因/蛋白的表达，实现维持软骨的稳态平衡，也进一步证实了自噬参与其中。此外，负载吡格列酮的SMSC EVs在增加自噬的同时，上调p62的表达，与预期相反，课题组正在进一步探讨p62通过Keap1-Nrf2信号通路调控OA软骨稳态平衡的分子机制。本研究将进一步阐明负载吡格列酮的SMSC EVs维持软骨稳态平衡的机理，为OA的精准治疗奠定基础。