L-dopa诱导PC12细胞凋亡及Bcl-2、Bax表达的改变

Study on the Induction of Apoptosis and the Expression of Bcl-2/Bax on Catecholaminergic PC12 Cells by L-dopa

目的 探讨 L-dopa治疗帕金森病 (PD)疗效减退的机制及其毒性作用机制。方法 以 PC1 2细胞为多巴胺神经元的细胞模型 ,利用 PI/HO3 3 3 4 2双染结合荧光显微镜技术、电镜技术、流式细胞术及免疫荧光技术检测不同浓度的 L-dopa对 PC1 2细胞的凋亡诱导作用及凋亡相关基因 Bcl-2、Bax表达的改变。结果 50、1 0 0、1 50μmol/L不同浓度 L-dopa处理组凋亡率分别为 1 2 .4 %、2 4 .4 %、3 7.2 % ;PI/HO3 3 3 4 2双染可区别凋亡、坏死和正常细胞 ,且可以见到染色质碎裂 ;电镜下可见早期凋亡细胞和晚期凋亡细胞。给予 L-dopa处理后 ,Bcl-2的表达量减少 ,与凋亡率呈显著负相关 ;Bax的表达量增加 ,与凋亡率呈显著正相关。结论 L-dopa诱导 PC1 2细胞凋亡且呈量效关系 ,提示 L-dopa可能是通过凋亡途径损害多巴胺神经元导致疗效减退 ,其机制可能是通过改变Bcl-2 /Bax的比值来介导细胞凋亡。

Objective To investigate the possible mechanism of eventual decline in effectiveness of L-dopa treatment, and the neurotoxicity mechanism of L-dopa.Methods Using PC12 cells as the model of dopaminergic neurons, and EM,FCM,PI/HO33342 double staining and immunofluorescence technology, the changes in ultrastructure, expression of the apoptosis-related gene Bcl-2 ,Bax and the apoptosis ratio of PC12 cells induced by L-dopa treatment were observed.Results Treatment of PC12 cells with concentrations of 50,100,150 μmol/L L-dopa for 24 hours revealed that the apoptosis ratio was 12.8%,24.4%,37.2% respectively; PI/HO33342 double staining could distinguish apoptotic cells as well as normal cells; Under EM we could identify the earlier and the later stage of apoptotic cells; Expression of the apoptosis-supression gene Bcl-2 was reduced while expression of the apoptosis-induction gene Bax was enhanced,the effect was dose dependent.Conclusions The induction of apoptosis in PC12 cells by L-dopa indicates that apooptosis may be the cause for dopaminergic neurons death after long-term treatment of L-dopa. It might be mediated by the changes in the expression of the apoptosis-related gene Bcl-2/Bax.