左旋多巴对C17.2神经干细胞的毒性作用及司来吉兰的神经保护作用

Toxicity of levodopa on C17.2 neural stem cell and neuroprotective action of selegiline against it

目的 探讨左旋多巴对C17.2神经干细胞的毒性作用和司来吉兰对其的拮抗作用。 方法 采用MTT法检测不同剂量左旋多巴对C17.2神经干细胞的毒性作用 ,用Brdu标记检测左旋多巴对细胞增殖的影响 ,用透射电镜、Annexin V染色荧光显微镜和流式细胞仪检测细胞凋亡 ,用WesternBlot检测Caspase3及其活性片段 ;相同条件下同时检测司来吉兰的保护作用。结果 MTT显示左旋多巴可使C17.2神经干细胞活性降低 ,与剂量相关 (P <0 .0 5 ) ,Brdu标记显示 2 0 0 μmol/L左旋多巴可使细胞增殖活力下降 ,与时间相关 (P <0 .0 5 ) ,作用 12及 2 4h后的透射电镜、Annexin V染色荧光显微镜和流式细胞仪可检测到凋亡细胞 ,WesternBlot显示Caspase 3表达量增加 ,并逐渐被切割成活性片段。司来吉兰能部分保护细胞免受左旋多巴的影响 (P <0 .0 5 ) ,并能部分抑制左旋多巴引起的细胞凋亡 (P <0 .0 5 )。 结论 大剂量左旋多巴对C17.2神经干细胞具有毒性作用 ,呈剂量和时间依赖性。毒性剂量的左旋多巴可通过抑制DNA合成影响细胞增殖 ,并通过活化Caspases 3促进细胞凋亡 ,司来吉兰可部分拮抗上述作用。

Objective To investigate the toxicity of levodopa on C17.2 neural stem cell and neuroprotective action of selegiline against it. Methods The possible cytotoxicity of levodopa at different dosages on C17.2 neural stem cells and the neuroprotective action of selegiline were determined by MTT assay. Cell proliferation was assayed by Brdu labeling and the apoptosis was detected by electronmicroscopy, Annexin-V-FLUOS staining and flow cytometry. The expression of caspase-3 and the cleavage of Caspase-3 were measured by Western Blot. Results Levodopa induced a concentration- and time-dependent decrease in cell viability and proliferation. There were some apoptotic cells at 12 and 24 h following exposure to levodopa at 200 μmol/L. The elevated Caspase-3 protein level and the cleaved Caspase-3 were demonstrated in C17.2 neural stem cells exposed to levodopa. These alterations could be partly inhibited by selegiline. Conclusion Levodopa at higher concentration ( ≥200 μmol/L) was neurotoxic to C17.2 neural stem cells through the inhibition of DNA synthesis and cell proliferation. The activation of Caspase-3 protease may contribute to the mechanism by which levodopa induced cell apoptosis. Selegiline, an anti-Parkinson drug,could partly block levodopa-induced cytotoxicity to serve as a neuroprotective factor.