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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 ,也不通过升高胞内cAMP水平来实现
Objective To study whether theophylline has a protective effect on the apoptosis of cerebellar granule neurons induced by repolarizing potassium ion (5 mmol·L-1 KCl, also known as hypokalemia). Methods Cultured rat cerebellar granule neurons were cultured in vitro. The cell apoptosis was detected qualitatively and quantitatively. ① Cell viability was detected by fluorescein staining (FDA), and morphological changes of nuclei were analyzed by Hoechst 332 5 8 staining. (2) DNA was analyzed by agarose gel electrophoresis Fracture; ③ immunofluorescence assay intracellular cAMP levels. Results Theophylline increased the survival rate of cerebellar granule neurons cultured in hypokalemia, and caused the disappearance of nuclear condensation, aggregation and rupture caused by hypokalemia. Low potassium led to obvious “ladder-like” DNA electrophoresis pattern of neurons Theophylline caused this phenomenon to disappear; the intracellular calcium release blockers, L-type calcium channel blockers and NMDA receptor blockers could not inhibit the protective effect of theophylline on neurons; Theophylline had no effect on intracellular cAMP level Obvious impact. Conclusion Theophylline has a protective effect on the apoptosis of cerebellar granule neurons induced by repolarization and concentration of potassium ions. This effect is not dependent on intracellular calcium concentration nor is it achieved by increasing intracellular cAMP level