诱导性多能干细胞源性MSCs通过线粒体输送对心跳骤停后脑损伤的保护

Mitochondrial dysfunction and energy deficiency resulted from it play an important role in the cerebral injury after cardiac arrest(CA). However, there is no effective treatment to promote cerebral function via improving the function of mitochondria. Bone marrow derived mesenchymal stem cells (MSC) protect against acute lung injury by mitochondrial transfer to injured epithelial cells through Cx43-dependent gap junctional channels. Recently, MSC was succefully induced from induced pluripotent stem cell (iPSC). We found that iPSC-MSC significantly improved cerebral function in a rat model of CA. Furthermore, using coculture model of iPSC-MSC and neuroal cell line, we observed formation of tunneling nanotubes (TNTs) between MSC and neuroal cell, and there was exchange of mitochondria between two types of cells via TNTs. In which, Cx43 played the important role. We next hypothesize that (1) iPSCMSC has the potential to protect neurons via transfering fresh mitochondrias after CA; (2) Cx43 transfection to iPSC-MSC improves the above effects. In this project, we induce a coculture model of iPSC-MSC and neuronal cell line and primary neurons and a rat model of CA. We aim to investigate whether Ipsc-MSC transports the fresh mitochondrias, recovers energy metabolism,inhibits oxidative stress and promotes cerebral function; to examine whether Cx-43-transfected iPSC-MSC improves the above effects and further prevents cerebral function. The scientist who first reported iPS won the Nobel Prize in Physiology or Medicine in 2012. This study is timely and may provide useful information to the mechanism of neuroprotection of iPSCMSC on cerebral injury after CA.

线粒体结构功能障碍以及因此而致的能量匮乏是导致心跳骤停(CA)后脑损伤的核心环节，目前尚无针对此靶点的有效措施。骨髓间充质干细胞（MSC）可通过Cx43蛋白形成的连接通道向上皮细胞转移线粒体而减轻肺损伤。我们报道了诱导性多能干细胞（iPSC）来源的MSC可显著提高CA大鼠的脑功能。我们还发现iPSC-MSC与神经细胞间可形成连接通道并输送线粒体、抑制凋亡；而起桥梁作用的正是Cx43。因此我们设想iPSC-MSC可通过"线粒体输注"的方式向受损细胞提供新鲜线粒体而防治脑损伤，而上调Cx43可强化此作用。本课题拟通过细胞共培养和大鼠CA模型，观察iPSC-MSC能否发挥输送新鲜线粒体、恢复能量代谢、拮抗氧化应激、改善脑功能的作用；并转染Cx43以寻求更有效的干细胞治疗技术。iPS是个突破性的科学发现，iPSC-MSCs具有强大应用优势，本研究有助阐明其脑保护的作用机制，并为其临床应用提供依据。

线粒体结构功能障碍以及因此而致的能量匮乏是导致心跳骤停(CA)后脑损伤的核心环节，本研究尝试从干细胞转移输注线粒体的角度，探讨干细胞能否在体内体外发挥抗缺氧损伤的作用。.结果发现，CoCl2呈浓度依赖性地降低PC12细胞的存活率，线粒体结构显著退变。iPSC-MSCs与PC12细胞共培养时均能与iPSC-MSCs间形成隧道纳米管（TNTs）结构，并通过TNTs将线粒体转移到PC12细胞中。PC12细胞缺氧损伤时iPSC-MSCs的线粒体转移率明显升高可能与TNTs形成增多有关。阻断TNTs的形成可以抑制细胞间的线粒体转移。iPSC-MSCs通过TNTs向PC12细胞转移线粒体形成线粒体替代，对抗CoCl2对PC12细胞线粒体的结构和功能损伤，减轻氧化应激损伤、改善能量代谢，从而对缺氧诱导的细胞凋亡发挥抑制作用。正常组及损伤组PC12细胞胞浆中均可见Cx43蛋白表达，Cx43蛋白参与了TNTs的形成，但以Cx43为基础的GJCs在iPSC-MSCs向PC12细胞的线粒体转移中不起主要作用，这提示Cx43只是参与线粒体转移的因素之一有关。使用细胞松弛素CytoD可以显著减少iPSC-MSCs向PC12线粒体转移，而加入C43阻断剂Gap26则无明显影响就分别印证了以上结果。.在体内实验中，多种干细胞株型对心跳骤停后脑细胞损伤有保护作用。结果显示，干细胞治疗组的海马神经元内有来自于干细胞的线粒体。治疗组的线粒体结构基本完好，线粒体功能指标受损较轻、而氧化应激分子表达量显著降低。这表明，干细胞在体内也可以通过线粒体转移替代、减少神经元凋亡而发挥对心跳骤停后脑损伤的保护作用。.本研究在证明干细胞治疗对心跳骤停后全脑缺氧损伤有治疗作用的基础上，从线粒体转移替代的角度首次探讨了其具体机制，有助于更大程度上发挥干细胞治疗的作用，为其临床使用提供理论基础和科学依据。