Bone marrow MSCs and H2O2-injured tenocytes were co-cultured, and mitochondrial transfer was visualized by MitoTracker dye staining. Mitochondrial function, including mitochondrial membrane potential, oxygen consumption rate, and adenosine triphosphate content, was quantified in sorted tenocytes. Tenocyte proliferation, apoptosis, oxidative stress, and inflammation were analyzed. Furthermore, a collagenase type I-induced rat AT model was used to detect mitochondrial transfer in tissues and evaluate Achilles tendon healing.
MSCs successfully donated healthy mitochondria to in vitro and in vivo damaged tenocytes. Interestingly, mitochondrial transfer was almost completely blocked by co-treatment with cytochalasin B. Transfer of MSC-derived mitochondria decreased apoptosis, promoted proliferation, and restored mitochondrial function in H2O2-induced tenocytes. A decrease in reactive oxygen species and pro-inflammatory cytokine levels (interleukin-6 and -1β) was observed. In vivo, mitochondrial transfer from MSCs improved the expression of tendon-specific markers (scleraxis, tenascin C, and tenomodulin) and decreased the infiltration of inflammatory cells into the tendon. In addition, the fibers of the tendon tissue were neatly arranged and the structure of the tendon was remodeled. Inhibition of mitochondrial transfer by cytochalasin B abrogated the therapeutic efficacy of MSCs in tenocytes and tendon tissues.
MSCs rescued distressed tenocytes from apoptosis by transferring mitochondria. This provides evidence that mitochondrial transfer is one mechanism by which MSCs exert their therapeutic effects on damaged tenocytes.
方法:将骨髓间充质干细胞与H2O2损伤的肌腱细胞共培养,线粒体转移通过MitoTracker染料染色可视化。线粒体功能,包括线粒体膜电位,耗氧率,和三磷酸腺苷含量,在分选的肌腱细胞中定量。肌腱细胞增殖,凋亡,氧化应激,并对炎症进行了分析。此外,使用胶原酶I型诱导的大鼠AT模型来检测组织中的线粒体转移并评估跟腱愈合。
结果:MSCs成功地将健康的线粒体捐献给体外和体内受损的肌腱细胞。有趣的是,通过与细胞松弛素B共同治疗,线粒体转移几乎完全被阻断。MSC衍生的线粒体的转移减少了细胞凋亡,促进扩散,并恢复H2O2诱导的肌腱细胞的线粒体功能。观察到活性氧和促炎细胞因子水平(白介素6和-1β)的降低。在体内,MSCs的线粒体转移改善了肌腱特异性标志物的表达(巩膜,生腱C,和腱调节蛋白)并减少了炎症细胞向肌腱的浸润。此外,肌腱组织的纤维排列整齐,肌腱结构重塑。细胞松弛素B对线粒体转移的抑制消除了MSC在肌腱细胞和肌腱组织中的治疗功效。
结论:MSCs通过转移线粒体来拯救受损的肌腱细胞免于凋亡。这提供了线粒体转移是MSC对受损肌腱细胞发挥治疗作用的一种机制的证据。