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目的:研究抗抑郁药地昔帕明(Des)对胶质瘤C6细胞的凋亡诱导作用以及对凋亡关键效应分子caspase3和凋亡早期信号[Ca~(2+)]_i的调控作用.方法:采用流式细胞术(FCM)和凝胶电泳观察Des对C6细胞凋亡的DNA裂解作用,RT-PCR分析。caspase 3基因的表达以及激光扫描共聚焦显微镜测量单个活细胞[Ca~(2+)]_i浓度.结果:Des(10,20,40μmol/L)处理C6细胞24 h后,FCM图的G_1峰左侧出现凋亡特征性亚二倍体细胞峰,凋亡细胞百分率分别为5.2％,21.9％和 41.9％.同时,凝胶电泳显示典型的DNA“梯带”.DeS 20 μmol/L处理C6细胞24 h可明显增强caspase 3基因的表达,而未经Des处理的C6细胞则检测不到caspase 3基因的表达.此外,Des 40 μmol/L可使 C6细胞[Ca~(2+)]_i迅速升高并维持超过28 min,而钙螯合剂依他酸可显著降低C6细胞[Ca~(2+)]_i增高幅度,提示Des致C6细胞[Ca~(2+)]_i增高主要与细胞外钙内流有关.结论:Des诱导C6胶质瘤细胞凋亡可能与caspase 3基因表达的上调以及细胞内钙稳态的失衡有关.
AIM: To investigate the apoptosis-inducing effect of antidepressants desipramine on C6 glioma cells and its role in the regulation of the key apoptotic effector caspase3 and early apoptotic signal [Ca2 +] i. Methods: Flow cytometry (FCM) and gel electrophoresis were used to observe the effect of Des on DNA cleavage of C6 cells by RT-PCR. The expression of caspase 3 and the concentration of [Ca 2+] i in single living cells were measured by laser scanning confocal microscopy.Results: After treatment with Des (10, 20, 40 μmol / L) for 24 h, The apoptotic sub-diploid cell peak appeared on the left, with the percentage of apoptotic cells being 5.2%, 21.9% and 41.9%, respectively.At the same time, gel electrophoresis showed a typical DNA “ladder.” DeS 20 μmol / L treatment C6 However, the expression of caspase 3 gene was not detected in the untreated C6 cells after treatment with Des 40 μmol / L for 24 h, and the [Ca 2+] i And increased rapidly for more than 28 min. However, the calcium chelator, ethacrynic acid, significantly decreased the increase of [Ca2 +] i in C6 cells, suggesting that the increase of [Ca2 +] i in C6 cells induced by Des Extracellular calcium influx.Conclusion: Des induced C6 cell apoptosis may be related to the up-regulation of caspase 3 gene expression and intracellular calcium homeostasis.