从转录环节研究喜树碱诱导神经元凋亡的内在机制

The Inherent Mechanism of Neurotoxic Activity of Camptothecin from DNA Transcription Stage in Cultured Cerebellar Neurons

目的:从DNA转录环节探讨喜树碱对原代培养小鼠神经元毒性作用的内在机制。方法:利用原代培养的小鼠皮层神经元,分别用喜树碱和喜树碱联合转录抑制剂5,6-二氯-1-β-呋核亚硝脲-苯并咪唑(5,6-dichloro-1-β-D ribofuranosyl-benzimidazole,DRB)干预后,CCK-8检测各组神经元活性变化,然后通过TUNEL染色法、γ-H2AX免疫荧光标记、流式细胞法,检测神经元DNA损伤及凋亡情况。结果:喜树碱可明显抑制神经元的活性,与对照组比较,喜树碱组神经元γ-H2AX和TUNEL阳性染色细胞明显增多;与喜树碱组比较,喜树碱联合DRB组γ-H2AX和TUNEL阳性染色细胞明显减少。流式细胞仪检测显示,喜树碱联合DRB剂组神经元晚期凋亡的发生率较喜树碱组明显降低,而正常未受损细胞比例明显上升。结论:转录抑制剂DRB可有效减轻喜树碱诱导的神经元损伤和凋亡,提示喜树碱的神经元毒性机制可能与转录环节有关。

Objective： To study neurotoxicity mechanism of camptothecin through DNA transcription in cultured cerebellar neurons in mice. Methods：The primary cerebellar neurons were administrated by camptothecin or camptothecin and DRB respectively. Then, CCK-8 assay, TUNEL staining, γ-H2 AX immunofluorescence labeling and flow cytometry were tested. Results： Compared with the control group, neurons in camptothecin group showed more γ-H2 AX and TUNEL positive staining; Compared with the camptothecin group,neurons in camptothecin and DRB group showed much less γ-H2 AX and TUNEL positive staining. Flow cytometry showed increasing of the late apoptosis rate in the camptothecin group, however, decreasing of the late apoptosis rate in the camptothecin and DRB group. Conclusion：DRB, a transcription inhibitor, could effectively reduce the neuronal injury and apoptosis induced by camptothecin, showing that camptothecin-induced neurotoxicity may be related with the DNA transcription.