【摘要】 以培养8～10天的大鼠海马神经元为对象,选择CalciumOrangeAM和DAF-FMdiacetate为Ca2+和一氧化氮(NO)的荧光指示剂,建立了基于激光扫描共聚焦显微技术的细胞内Ca2+和NO双标记检测方法.此方法对Ca2+和NO进行分步染色,然后应用激光扫描共聚焦显微镜(LSCM)的双轨迹(TwoTrack)模式,通过快速切换激光实现对细胞内Ca2+和NO的同时检测.实验结果显示,两种染料之间无串扰现象;在N-甲基-D-天冬氨酸(NMDA)刺激下,海马神经元胞内Ca2+快速升高,随后达到平台期并有波动,NO则稳定持续升高,这些变化过程与单标记的结果一致;双标记层切序列图像显示细胞内Ca2+和NO都较集中分布于细胞中部,但在细节上两者的分布存在差异.此双标记方法能同时检测培养的海马神经元胞内Ca2+和NO,为研究神经元胞内Ca2+和NO的相互调控作用提供了一种新的手段.更多还原

【Abstract】 Choosing 8~10 days cultured hippocampal neurons of SD rat, using Calcium Orange AM and DAF-FM diacetate as the fluorescent indicators of intracellular Ca2+ and nitric oxide(NO), simultaneous detection of intracellular Ca2+ and NO was proposed by double-label method on laser scanning confocal microscope (LSCM). The dyeing process includes two steps and "Two Track" mode of LSCM is applied to realize simultaneous detection of intracellular Ca2+ and NO through quickly switching excitation wavelengths. The experiment results show that there is no cross talk between two dyes and the double-label method can reveal the changes of intracellular Ca2+ and NO concentrations under the stimulation of N-methyl-D-aspartate (NMDA), quite consistent with the results of respective single-label experiments. The analysis of slicing image sequences of double-labeled neurons shows that both Ca2+ and NO are mainly located in the center area of cell bodies, while their distribution details are different. The results suggest that the double-label method can simultaneously detect the intracellular Ca2+ and NO in cultured hippocampal neurons and thus provide an approach to investigate the roles of Ca2+ and NO in neurons as well as the interaction between them.更多还原