蛋白酶体抑制剂诱导多巴胺能神经元重启细胞周期的作用及机制

目的研究蛋白酶体抑制剂诱导多巴胺能神经元重启细胞周期的作用及机制。方法用蛋白酶体抑制剂lactacystin处理神经元样分化的PC12细胞,四甲基偶氮唑盐(MTT)法检测细胞活力,流式细胞仪检测早期凋亡细胞和细胞周期分布情况,RT-PCR检测细胞周期调控因子表达水平。结果经lactacys-tin处理后细胞活力呈剂量依赖性下降,出现早期凋亡细胞,随着处理时间的延长,细胞凋亡率逐渐增加;细胞周期中G0/G1期细胞减少,S期和G2/M期细胞增多;CyclinA和CyclinD1mRNA表达水平逐渐升高而Cy-clinB1mRNA表达水平无明显改变。结论蛋白酶体功能障碍通过上调细胞周期调控因子的过度表达而重新启动多巴胺能神经元的细胞周期,并诱发多巴胺能神经元细胞凋亡,产生神经元变性损伤。

The Effect and mechanism of proteasome inhibitor induced cell cycle reentry on dopaminergic neurons

Objective To investigate the effect and the mechanism of proteasome inhibitor induced cell cycle reentry on dopaminergic neurons.Methods PC12 cells as dopaminergic neurons, differentiated by nerve growth factor, were treated by proteasome inhibitor, lactacystin.Cell viability was measured by MTT, Flow cytometry was used to detect cell apoptosis and the changes of cell cycle, the expressions of cell cycle regulator Cyclin D1, Cyclin A and CyclinB1 mRNA were assayed by RT-PCR.Results After treated with lactacystin, the cell viability declined in a concentration-dependent manner. The early sign of apoptosis was found with flow cytometry, and the apoptosis ratio increased in a time-dependent manner. The percentage of cells in the G0/G1 phase decreased and that in the S and G2/M phase increased. This was associated with the increased mRNA levels of CyclinA and CyclinD1, while the mRNA level of CyclinB1 did not change.Conclusions The dysfunction of ubiquitin-proteasome system might induce cell apoptosis of dopaminergic neuron by up-expression of cell cycle regulator and reentry of cell cycle, which contributes to the effect of neurodegenerative injury.