细胞内游离Ca^(2+)在发育中大鼠皮层神经元无镁诱导惊厥性损伤中的作用  

Effect of intracellular-free Ca^(2+) concentration on transient magnesium-free treatment induced epileptic injury in developing cortical neurons of rats

作　　者：曹海燕[1] 姜玉武[1] 何其华[2] 陈跃[2] 袁兰[2] 吴希如[1] 

机构地区：[1]北京大学第一医院儿科,北京100034 [2]北京大学医学部医药卫生分析中心

出　　处：《北京大学学报（医学版）》2003年第5期466-470,共5页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金 ( 3 990 0 160 ;3 0 2 70 44 8)~~

摘　　要：目的 :观察细胞内游离Ca2 + ([Ca2 + ]i)在培养的不同发育阶段皮层神经元无镁诱导惊厥性损伤中的作用 ,探讨惊厥性脑损伤年龄依赖性的可能机制。方法 :体外培养 6d、17d的胚胎大鼠皮层神经元用无镁细胞外液处理 3h ,或于无镁处理前用NMDA(N 甲基 D 门冬氨酸 )受体拮抗剂或Ca2 + 通道阻滞剂预处理 ,用MTT代谢率测定的方法检测神经元损伤 ,以Fluo 3作标记用激光共聚焦显微镜扫描的方法检测 [Ca2 + ]i。结果 :体外培养 6d、17d的神经元单纯无镁组MTT代谢率较同期对照组降低。应用MK 80 110 μmol·L-1、AP 5 5 0 μmol·L-1、尼莫地平 10 μmol·L-1预处理后再给无镁处理 ,培养 6d、17d的神经元MTT代谢率均不同程度高于同期单纯无镁组。培养 6d、17d的神经元相对荧光强度之间差异有显著性 ,两者与基线荧光强度比较差异亦有显著性。应用上述各种拮抗剂后 ,[Ca2 + ]i 改变的峰值均明显低于同期单纯无镁组。结论 :在体外不同发育阶段的神经元 ,短暂无镁处理诱导惊厥样放电所引起的神经元线粒体功能损伤以及 [Ca2 + ]i 改变程度不同。这种 [Ca2 + ]i 改变的年龄依赖性可能是惊厥导致神经元损伤的年龄依赖性的机制之一。NMDA受体 Ca2 + 通道激活是导致这种 [Ca2 +Objective: To investigate the effect of intracellular free Ca 2+ concentration ([Ca 2+ ] i ) on injury following transient Mg 2+ free treatment in vitro in developing cortical neurons. Methods: Embryo cortical neurons of rats cultured for 6 d and 17 d were directly exposed to Mg 2+ free media, or pretreated with NMDA receptor antagonists or calcium channel antagonist before being exposed to Mg 2+ free media. MTT assay was used to study the injury of neurons. [Ca 2+ ] i were measured using fluo 3, a fluorescent calcium sensitive dye and laser scanning confocal microscope, and calculated by the fluorescent intensity. Results:Compared with control, MTT conversion rates decreased after transient (3 h) Mg 2+ free treatment in neurons cultured for 6 d and 17 d in vitro, (59.1±6.87)% and (51.2±5.90)%, respectively. In neurons pre and co treated with 10 μmol·L -1 MK 801, 50 μmol·L -1 AP 5 and 10 μmol·L -1 nifedipine, MTT conversion rates were higher than those of neurons with only Mg 2+ free treatment. Peak values of [Ca 2+ ] i in neurons cultured for 6 d and 17 d were 2.4±0.23 and 3.2±0.32, respectively Peak value of neurons 17 d in vitro was significantly higher than that of neurons 6 d in vitro (P <0.05). In neurons pre and co treated with MK 801, AP 5 and nifedipine, [Ca 2+ ] i were lower than those of neurons with only Mg 2+ free treatment. Conclusion: Neuronal injury and [Ca 2+ ] i changes following Mg 2+ free treatment induced seizure were different between neurons 6 d and 17 d in vitro . It suggested that the age dependent [Ca 2+ ] i changes might play a role in an age dependent manner of injury following Mg 2+ free treatment induced seizure. NMDA receptor Ca 2+ pathway activation was crucial in the [Ca 2+ ] i change and the cellular injury induced by Mg 2+ free treatment.

关 键 词：钙 代谢 神经元 惊厥 大脑皮质 培养的细胞 

分 类 号：R742.1[医药卫生—神经病学与精神病学]