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目的在细胞水平研究腺苷对原代培养的SD大鼠海马神经元内钙离子振荡的具体影响,并探讨其可能的作用机制,从而为腺苷的抗癫痫作用提供实验依据。方法本实验以SD大鼠新生48h内的乳鼠作为取材对象,以胰酶消化加巴氏管机械吹打的方法 ,经条件培养基纯化建立原代培养海马神经元的研究模型。选取7～9d的细胞,应用激光扫描共聚焦显微镜结合Fluo-3/AM标记技术,利用细胞外灌流方式给予腺苷的方法 ,检测腺苷干预前后钙离子振荡的相关指标。结果原代培养的SD大鼠乳鼠的海马神经元在7～9d发育基本成熟后可以观察到神经元内自发同步的钙离子振荡。给予腺苷后,发现Ade能抑制成熟海马神经元自发同步钙离子振荡的频率和幅度:频率从加药前的(0.020±0.003)Hz减少到(0.005±0.001)Hz,幅度从加药前的1.87±0.17下降到1.10±0.07,且这种抑制作用对于腺苷的浓度水平有一定的要求性。用高钾作为去极化刺激模拟癫痫状电活动时,钙离子震荡的频率加快,幅度增大,而腺苷对高钾诱发钙离子振荡的频率和幅度表现出了显著抑制作用。结论在体外培养海马神经元的研究中发现,腺苷对海马神经元内钙离子振荡具有显著的抑制作用,这可能是腺苷作为内源性物质发挥抗癫痫作用的机制之一。
Objective To study the effect of adenosine on calcium oscillations in hippocampal neurons of primary cultured SD rats at the cellular level and to explore its possible mechanism of action so as to provide an experimental basis for the antiepileptic effect of adenosine. Methods In this study, the neonatal SD rats born within 48h of the fetus as a drawing object, with pancreatin digestion plus Papaniculum tube method of mechanical blow through the conditioned medium to establish a primary cultured rat hippocampal neuronal model. The cells of 7-9 days were selected, and the relevant indicators of calcium oscillation before and after adenosine intervention were detected by laser scanning confocal microscopy combined with Fluo-3 / AM labeling and adenosine administration by extracellular perfusion. Results The hippocampal neurons of primary cultured SD rats could spontaneously synchronize calcium oscillations in neurons after 7 ~ 9 days of development. After administration of adenosine, Ade was found to inhibit the frequency and amplitude of spontaneous synchronous calcium oscillations in mature hippocampal neurons: the frequency was reduced from (0.020 ± 0.003) Hz before administration to (0.005 ± 0.001) Hz, 1.87 ± 0.17 decreased to 1.10 ± 0.07, and this inhibitory effect on adenosine concentration levels have certain requirements. When potassium is used as a depolarization stimulator to simulate epileptic electrical activity, the frequency of calcium oscillations is accelerated and the amplitude is increased. However, adenosine exerts a significant inhibitory effect on the frequency and amplitude of calcium oscillations induced by high potassium. Conclusion In vitro studies of hippocampal neurons showed that adenosine had a significant inhibitory effect on calcium oscillations in hippocampal neurons, which may be one of the mechanisms by which adenosine exerts antiepileptic effect as an endogenous substance.