茶碱对低钾诱导的小脑颗粒神经元凋亡的保护作用

The protective effect of theophylline against apoptosis induced by depriving of depolarization in cerebellar granule neurons

目的 研究茶碱对复极化浓度钾离子 (5mmol·L-1KCl,又称低钾 )诱导的小脑颗粒神经元凋亡是否具有保护作用。方法 体外培养大鼠小脑颗粒神经元 ,定性定量检测细胞凋亡 :①用FDA (fluoresceindiacetate)染色检测细胞存活率 ,用Hoechst 332 5 8染色 ,分析细胞核的形态变化 ;②琼脂糖凝胶电泳分析DNA断裂 ;③免疫荧光法检测细胞内cAMP水平。结果 茶碱使低钾培养的小脑颗粒神经元的存活率增加 ,使低钾引起的细胞核固缩、凝聚和断裂现象消失 ;低钾使神经元的DNA电泳图谱出现明显的“梯子状” ,而茶碱使此现象消失 ;敏感性内钙释放阻断剂、L 型钙通道阻断剂及NMDA受体阻断剂均不能抑制茶碱对神经元的保护作用 ;茶碱对细胞内cAMP水平没有明显的影响。结论 茶碱对复极化浓度钾离子诱导的小脑颗粒神经元凋亡具保护作用 ,此作用不依赖胞内钙离子浓度 。

AIM To study whether theophylline can prevent cerebellar granule neurons from apoptosis induced by depriving of depolarization. METHODS The following methods were used to investigate the protective effects of theophylline on cultured cerebellar granule neurons in low K + medium:①The neurons were stained with fluorescein diacetate to observe their survival rate or stained with Hoechst 33258 to analyze their nuclear morphology.②Internucleosomal DNA fragmentation of neurons was analyzed with agarose gel electrophoresis. ③Intracellular cAMP levels were determined by means of radioimmunoassay kits. RESULTS ①Theophylline increased the survival rate of cultured cerebellar granule neurons in low K + medium in a dose dependent manner and prevented the neuronal nuclei from shrinkage, condensation and cleavage induced by depriving of depolarization. ②Theophylline prevented neuronal nuclear DNA fragmentation induced by depriving of depolarization. ③The protective effect of theophylline was not affected by inhibitors of ryanodine sensitive Ca 2+ release,nor was it inhibited by L type channel blockers and NMDA receptor blocker MK801. ④Theophylline did not influence the intracellular cAMP levels in depolarization deprived cerebellar granule neurons. CONCLUSION Theophylline can prevent cerebellar granule neurons from apoptosis induced by depriving of depolarization and this effect is not dependent on intracellular Ca 2+ or cAMP levels.