三种组织线粒体对小鼠原代神经元存活及轴突再生的影响

The effects of mitochondria derived from three tissues on the survival and axonal regrowth of primary mouse neurons

目的:比较研究肝脏、心肌和骨骼肌来源的线粒体对小鼠原代神经元存活及轴突再生的影响。方法:采用差速离心法分别提取C57BL/6小鼠肝脏线粒体、心肌线粒体和骨骼肌线粒体。分离和培养孕14~17 d的C57BL/6胎鼠大脑皮质神经元,培养7 d后进行划痕损伤,然后分别加入等量的三种组织来源线粒体进行共培养。采用末端脱氧核苷酸转移酶介导的dUTP原位缺口末端标记法(TUNEL)、活细胞碘化丙啶(PI)标记观察神经元死亡。采用Western Blot和免疫细胞化学染色检测神经元中生长相关蛋白43(GAP-43)的表达。采用分光光度计测量线粒体体复合体酶活性和ATP水平。结果:TUNEL染色、PI染色显示:三种线粒体处理后,肝脏线粒体处理组的神经元死亡最少,提示肝脏线粒体的促神经元存活能力最强。肝脏线粒体处理组神经元GAP-43的表达水平最高。进一步分析发现:相同数量的肝脏线粒体产生更高水平的ATP。并且肝脏线粒体可显著促进神经元线粒体呼吸链复合体酶的活性。结论:与心肌线粒体、骨骼肌线粒体相比,肝脏线粒体具有更强大的促神经元存活和促轴突再生的能力,可用于体内线粒体移植修复神经损伤。

Objective:To compare the effects of three different exogenous mitochondria on neuronal survival and axonal regrowth.Methods:Mitochondria were isolated from liver,cardiac muscle and skeletal muscle by differential centrifugation.Primary neurons were isolated from the cerebral cortex of E14-E17 embryos.The cells were cultured for 7 days before scratching injury.Same amount of liver mitochondria,skeletal muscle mitochondria or cardiac mitochondria were added into culture medium.The cell death was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling(TUNEL)and live cell PI-labeling.The expression of growth-associated protein 43(GAP-43)was evaluated by immunocytochemistry and Western Blot.Spectrophotometer was used to measure the substrate of mitochondrial complex and ATP levels.Results:TUNEL and PI-staining showed that liver mitochondria were most effective in alleviating cell death.The data of GAP-43 levels showed that liver mitochondria had the strongest effect of stimulating the expression of CGAP-43.Further analysis showed highest ATP production from liver mitochondria,comparing with same amount of cardiac and skeletal muscle mitochondria.Further,adding exogenous liver mitochondria significantly increased the activity of mitochondria complex.Conclusion:In comparison with skeletal muscle mitochondria and heart mitochondria,liver mitochondria are most powerful in promoting neuronal survival and axonal regeneration in vitro,indicating that liver mitochondria could be used as an optimal transplant for repairing neural injury in vivo.