摘要
目的 观察线粒体功能失调在缺血再灌注后神经细胞凋亡中的作用。方法 ( 1)采用体外培养神经母细胞瘤细胞株N2a,模拟缺血再灌注 (先缺氧缺营养 90min ,然后正常培养不同时间 ) ;( 2 )采用琼脂糖凝胶电泳检测细胞凋亡情况 ;( 3)通过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 )Cas pase 3活性测定采用水解其可见光底物。结果 ( 1)N2a细胞缺血再灌注 12h即出现明显DNA片段化 ,2 4h更明显 ;( 2 )线粒体琥珀酸脱氢酶活性缺血再灌注 2 4h明显降低 ;跨膜电位在缺血再灌注 1h先短暂下降 ,3h明显升高 ,6h后降低 ,以后再没回升。缺血再灌注 3h细胞色素C开始释放 ,6h达到高峰 ,并持续到 2 4h ;( 3)Caspase 3活性在缺血再灌注 10h升高 ,2 4h达到高峰。线粒体通透性转换孔抑制剂cyclosporineA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 缺血再灌注诱导神经细胞凋亡存在caspase 3依赖性和非依赖性两条途径 。
Objective To observe the mechanism of neuron apoptosis undergoing ischemia reperfusion injury.Methods The following ways were used:(1)Culturing neuroblastoma cell line N2a in vitro and modeling ischemia reperfusion(firstly deprive of nutrition and oxygen for 90 minute,then reperfusion different times). (2)detecting cell apoptosis by agar gel electrophoresis and FCM(flow cytometry). (3)observing mitochondria function through MTT?cytochrome C release and monitoring trans membrane potential. (4)determining caspase 3 activity by catalysing visible substrate. Results (1)DNA fragmentation was observed after ischemia reperfusion 12 hours especially clean after 24 hours.(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 began from reperfusion 3 hours,reached peak at reperfusion 6 hours and maintained reperfusion 24 hours.(3)Caspase 3 activity 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 can completely reverse DNA fragmentation, but could not completely inhibit caspase 3 activity.Conclusions There were two apoptosis pathways in neuron, caspase 3 dependent and independent pathways.The roles of mitochondria dysfunction may early trigger apoptosis and amplify apoptosis signal.
出处
《卒中与神经疾病》
2004年第5期271-274,共4页
Stroke and Nervous Diseases
