【摘要】 目的观察线粒体功能失调在缺血再灌后神经元凋亡中的作用。方法(1)采用体外培养神经母细胞瘤细胞株N2a细胞,体外模拟缺血再灌注(缺氧90min,然后再灌不同时间);(2)采用琼脂糖凝胶电泳检测细胞凋亡情况;(3)MTT法、细胞色素C释放和跨膜压的改变判断线粒体的功能;(4)caspase-3活性测定采用水解其可见光底物。结果(1)N2a细胞缺血再灌12h即出现明显DNA片段化,24h更明显;(2)线粒体酶活性降低,跨膜电位在缺氧再灌1h先短暂下降再升高然后又明显降低,细胞色素C缺氧再灌3h开始下降,6h达到高峰,持续到24h(3)caspase-3活性在缺氧再灌10h升高,24h达到高峰,线粒体通透性转换孔形成抑制剂CsA只能抑制部分caspase-3的活性和DNA片段化改变;而caspase-8抑制剂虽不能完全抑制caspase-3的活性但能完全抑制DNA的片段化。结论N2a凋亡尚存在有caspase-3依赖性和非依赖性两条途径线粒体功能失调在缺氧再灌引起的N2a细胞凋亡过程中可能起到凋亡早期的启动和随后的信号放大作用。更多还原

【Abstract】 Objective To observe the role of mitochondria dysfunction in neuron apoptosis during ischemia-reperfusion injury.Methods (1)Culturing neuroblastoma cell line N2a in vitro and modeling ischemia-reperfusion (firstly deprive of nutrition and oxygen for 90 minute,then reperfusion for different time).(2)Detecting cell apoptosis by agar gel electrophoresis.(3) To observe mitochondria function through MTT,cytochrome C release and monitoring transmembrane potential. (4) Determining caspase-3 activity by catalysis visible substrate.Results (1)DNA fragmentation was observed after ischemia-reperfusion 12 hours,while 24 hours more clearly.(2)Mitochondria enzyme activities was reduced and trans-membrane potential firstly decreased after ischemia-reperfusion 1 hour,then sharply increased and reduced.Release of cytochrome C beginned from reperfusion 3 hours,reached a peal at reperfusion 6 hours and maintained reperfusion 24 hours.(3) Caspase3 activity was increased from reperfusion 10 hours to 24 hours.Moreover,Cyclosporine A,an inhibitor of mitochondria permeability transition pore,only partly inhibited caspase-3 activity and DNA fragmentation.Interestingly,caspase-8 inhibitor could completely reverse DNA fragmentation,but couldn’t completely inhibit caspase-3 activity.Conclusion There are two apoptosis pathways in neuron,caspase-3 dependent and independent pathways.The roles of mitochondria dysfunction may trigger apoptosis in the early period and amplify apoptosis signal.更多还原