突变型低氧诱导因子-1α修饰骨髓间充质干细胞来源的外泌体联合软骨再生支架治疗家兔早期软骨缺损

Effects of exosomes derived from mutant hypoxia inducible factor-1α modified bone mesenchymal stem cells on chondrocyte apoptosis and its role on the regeneration of early cartilage defects of the rabbits combined with porous scaffold

目的 探讨突变型低氧诱导因子-1α（HIF-1α）修饰的骨髓间充质干细胞（BMSCs）来源的外泌体（BMSC-Exo）对关节软骨损伤的修复作用.方法 BMSC和软骨细胞培养;超高速离心法提取突变型HIF-1 α修饰的BMSC分泌的外泌体（BMSC-Exomu）及正常BMSC分泌的外泌体（BMSC-Exonor）;噻唑蓝（MTT）法检测软骨细胞活性;Western blot分析BMSC-Exos调节细胞凋亡过程中的作用.构建早期软骨缺损动物模型,并分为4组：单纯缺损组、支架植入治疗组、支架＋BMSC-Exonor组、支架＋BMSC-Exomu组.术后6周取材,通过大体观察、苏木素-伊红（HE）染色、蕃红O-快绿染色,比较各组软骨缺损的修复效果.结果 MTT法与Western blot结果显示,同对照组比较,BMSC-Exonor能对TNF-α介导的软骨细胞凋亡发挥作用,差异有统计学意义（P=0.006）.而BMSC-Exomu效果更为显著（P=0.001）.动物实验发现,与其他各组比较,支架＋BMSC-Exomu组的软骨修复作用最强、膝关节炎的症状较其他组均有明显好转,其损伤面透明软骨形成最多,软骨基质分泌最多.结论 在TNF-α诱导的软骨细胞凋亡中,与BMSC-Exonor比较,BMSC-Exomu对软骨细胞具有更强的保护作用.BMSC-Exo的应用对于家兔早期软骨缺损具有较好的辅助治疗效果.

Objective Cartilage defects is one of the common joint diseases throughout the world.We hypothesized that exosomes may have supporting role in treating this disease.Methods In vitro,bone marrow mesenchymal stem cells （BMSCs） were transfected with or without mutant hypoxia inducible factor （HIF）-1α.Exosomes derived from both kinds of BMSCs （BMSC-Exonor or BMSC-Exomu） were isolated and identified.Chondrocytes were treated with tumor necrosis factor （TNF）-α,and BMSC-Exonor or BMSC-Exomu were also added at the same time.Proliferation,apoptosis,and the activity of mitogen-activated protein kinases （MAPKs） and protein kinase B （Akt） were examined.In vivo,BMSC-Exonor or BMSC-Exomu were combined with porous scaffold to repair cartilage defects,and the efficacy was assessed by histological examination.Results MTT assay and Western blot results showed that BMSC-Exonor could play a role in TNF-α-mediated chondrocyte apoptosis,the difference was statistically significant （P =0.006）,while BMSC-Exomu was more effective （P =0.001）.While in vivo,BMSC-Exomu improved regeneration of early cartilage defects of the rabbits combined with scaffold.Conclusion These findings highlight the supporting potential of BMSC-Exomu in repairing cartilage defects.Such exosomes combined with porous scaffold may represent a novel approach for the treatment of cartilage defects.