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（20，40，80 μM）醋酸铜诱导体外培养72 h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48 h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4 h 20 μM组开始升高，48 h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24 h 20 μM组才开始升高，激活的时间晚于caspase-8和caspase-9，48 h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。［中国当代儿科杂志，2009，11（11）：917-922］

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 copper 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.［Chin J Contemp Pediatr, 2009, 11 (11):917-922］