【作者】 杨帆；

【导师】 熊蠡茗；

【作者基本信息】 华中科技大学 ， 外科学（骨科）， 2022， 硕士

【摘要】 目的:细胞能量代谢障碍是压力等外界因素导致椎间盘退变的重要机制。以干细胞为基础的治疗已被证明有利于椎间盘的内源性修复,然而,其潜在机制尚未被完全阐明。本研究的目的是探究骨髓间充质干细胞(BMSCs)能否主动通过隧道纳米管(TNTs)介导的线粒体转运来挽救受损的髓核细胞(NPCs),并研究关键蛋白Miro1在这一过程中的调控作用。方法:将NPCs与BMSCs进行共培养,采用激光共聚焦扫描显微镜观察细胞间线粒体转运和TNTs的形成。通过检测线粒体膜电位、活性氧(ROS)、ATP、ADP、NAD+和NADH的水平来评估线粒体转运对受损NPCs线粒体功能的影响。通过检测细胞凋亡率和凋亡相关蛋白的变化水平来评估线粒体转运对受损NPCs凋亡的影响。利用携带Rhot1 sh RNA的腺病毒来减少BMSCs中Miro1的表达,探究Miro1在线粒体转运中的调节作用。结果:在共培养系统中,BMSCs将线粒体转运给了邻近的NPCs,此外,这些细胞间的线粒体转运方式为TNTs。在鱼藤酮诱导了NPCs的线粒体功能障碍后,NPCs可以从BMSCs中摄取更多的线粒体。TNTs介导的线粒体转运挽救了受损NPCs的线粒体功能障碍和凋亡。此外,Miro1的缺失减少了细胞间的线粒体转运,抑制了BMSCs对受损NPCs的挽救作用。结论:本研究揭示了BMSCs挽救受损NPCs的新机制,为研究细胞间相互作用在椎间盘内源性修复中的关键作用提供了坚实基础。更多还原

【Abstract】 Objectives:Cellular energy metabolism disorder is an important mechanism of intervertebral disc degeneration caused by compression and other external factors.Stem cell-based therapies have been shown to benefit endogenous disc repair.However,the underlying mechanisms have not been fully elucidated.The purpose of this study was to investigate whether bone marrow mesenchymal stem cells(BMSCs)could actively rescue impaired nucleus pulposus cells(NPCs)through mitochondrial transfer mediated by tunneling nanotubes(TNTs),and to investigate the regulatory role of the key protein Miro1 in this process.Methods:NPCs were cocultured with BMSCs.Confocal laser scanning microscopy was employed to observe the intercellular mitochondrial transfer and the formation of TNTs.The effects of mitochondrial transfer on mitochondrial dysfunctional NPCs were evaluated by measuring mitochondrial membrane potential,reactive oxygen species(ROS),ATP,ADP,NAD+ and NADH levels.Through detecting apoptosis rate and apoptosis-related proteins,the effects of mitochondrial transfer on apoptosis of damaged NPCs were assessed.Adenovirus carrying Rhot1 sh RNA to knockdown Miro1 expression in BMSCs were used to verify the functional role of Miro1 in mitochondrial transfer.Results:BMSCs donated mitochondria to adjacent NPCs in a coculture system.In addition,the means of mitochondrial transfer between these cells were intercellular TNTs.After rotenone-mediated induction of mitochondrial dysfunction in NPCs,NPCs could take up additional mitochondria from BMSCs.TNT-mediated mitochondrial transfer rescued NPCs from mitochondrial dysfunction and apoptosis.Furthermore,Miro1 depletion conferred a disadvantage to mitochondrial transfer and inhibited the rescue of NPCs with mitochondrial dysfunction.Conclusion:This study shed light on a novel mechanism by which BMSCs rescue NPCs from mitochondrial dysfunction and apoptosis,providing a solid foundation for studying the key role of intercellular interactions in endogenous disc repair.更多还原