Caspase-3,-8,-9在铜诱导大鼠原代皮层神经元凋亡中的作用研究

目的通过建立体外铜诱导神经元模型,观察醋酸铜对大鼠原代皮层神经元凋亡及活化caspase-3,caspase-8和caspase-9蛋白表达的影响,探索高浓度铜导致大鼠神经元损伤的作用机制。方法采用不同浓度(20,40,80μM)醋酸铜诱导体外培养72h的大鼠原代皮层神经元;MTT法检测神经元活力,Hoechst33258染色和流式Annexin-V/PI法检测神经元凋亡,Western blot法检测不同浓度和时间点活化的caspase-3、caspase-8和caspase-9蛋白表达。结果体外培养的原代皮层神经元经醋酸铜诱导48h后,凋亡率上升,细胞活力呈浓度梯度下降,活化的caspase-8和caspase-9在铜诱导4h20μM组开始升高,48h各浓度组表达最高,呈时间与浓度梯度依赖;活化caspase-3在铜诱导24h20μM组才开始升高,激活的时间晚于caspase-8和caspase-9,48h各浓度组表达最高。结论过量铜可导致体外培养大鼠原代皮层神经元发生凋亡,caspase-3、caspase-8和caspase-9级联反应在铜诱导皮层神经元凋亡过程中发挥重要调控作用。

Role of caspase-3,-8,and -9 in apoptosis of copper-induced primary cortical neurons

Objective This study aimed to investigate the mechanism of primary cortical neuron injury induced by high concentrations of copper by observing the effect of aceticum culture-medium on apoptosis of rat primary cortical neurons and expression of cleaved caspase-3, caspase-8 and caspase-9. Methods Primary cortical neurons were cultured for 72 hrs and then exposed to different concentrations of aceticum culture-medium (20, 40 and 80 μM). The viability of neurons was detected by the MTT method. Apoptosis was observed by Hoechst33258 and flow cytometry Annexin-V/PI. Expression of caspase-3, caspase-8 and caspase-9 was measured by Western blot. Results Following incubation with aceticum culture-medium, apoptosis of neurons was induced. The viability of neurons was remarkably reduced and the rate of apoptosis was tremendously increased in a concentration-dependent manner. Caspase-8 and caspase-9 were activated in 20 μM of copper aceticum culture-medium 4 hrs after incubation and peaked at 48 hrs in various concentrations of eopper aceticum culture-medium, presenting with a time and concentration-dependent manner. The activated caspase-3 was observed in 20 μM of copper aceticum culture-medium 24 hrs after incubation, which was later than the activated caspase-8 and caspase-9. Caspase-3 expression reached a peak 48 hrs in various concentrations of copper aceticum culture-medium, presenting with a time and concentration-dependent manner. Conclusions The apoptosis of primary cortical neurons can be induced by copper. Caspase-3, caspase-8 and caspase-9 cascade reaction may involve in the apoptosis of copper-induced rat primary cortical neurons.