帕金森病细胞模型的建立及鱼藤酮对多巴胺能神经元的毒性作用

目的：建立帕金森病细胞模型，研究鱼藤酮对多巴胺能神经元的毒性作用及机制。方法：PC12细胞诱导分化成多巴胺能神经元，加入不同浓度鱼藤酮（0、10、25、50、75和100 nmol•L^-1），观察细胞形态改变；鱼藤酮处理PC12细胞24、48和72 h，MTT法检测细胞活性；免疫组化和免疫荧光法观察α-synuclein 在胞内聚集情况；AO/EB双染检测细胞凋亡。结果：神经生长因子（NGF）诱导后PC12细胞形状不规则，突起增多变长，细胞间连接增多；染毒后细胞突起结构逐渐消失，细胞体积变小、形态变圆。50 nmol•L^-1鱼藤酮作用24 h后PC12细胞活力开始低于对照组（P<0.05），随着浓度增大，细胞活力进一步下降，呈浓度依赖性（P<0.01）；随着作用时间延长，细胞活力亦逐渐下降，不同时间组间比较差异有显著性(P<0.01)。免疫组化结果显示，胞浆中出现棕色的类圆形α-synuclein染色阳性颗粒；免疫荧光显示，胞浆中有α-synuclein标记的强绿色荧光的蛋白聚集。染毒细胞逐渐呈现出早期凋亡细胞、晚期凋亡细胞和坏死细胞状态。结论：鱼藤酮对多巴胺能神经元有毒性作用，可形成类包涵体样蛋白聚集并诱导细胞凋亡。α-synuclein 代谢异常及细胞凋亡可能在鱼藤酮所致的帕金森病发病机制中发挥重要作用。

Establishment of cell model of Parkinson's disease and toxic effect of rotenone on dopaminergic neurons

Objective To establish cell model of Parkinson's disease(PD)and to approach the toxic effect of rotenone on dopaminergic neurons and its mechanism.Methods PC12 cells were differentiated into dopaminergic neurons and treated with various concentrations of rotenone.The morphological changes of PC12 cells were observed after treated with rotenone(0,10,25,50,75,and 100 nmol·L-1)for 24,48 and 72 h,the cell viability was measured by MTT assay.Immunohistochemistry and immunofluorescence were used to observe the accumulation of α-synuclein in cytoplasm.AO/EB double staining was also adopted to test apoptosis.Results After differentiation PC12 cells shaped irregularly with big and long ecptomas and multiple cell conjunctions.After the treatment of rotenone cell ecptomas vanished gradually and cell bodies became smaller and smoother.The cell viability began to decline significantly at a concentration of 50 nmol·L-1 for 24 h(P<0.05),and as concentration increased,the cell viability declined in a dose-dependent manner(P<0.01).As time prolonged,the cell viability behaved the same way.There was a significant difference between each time groups(P<0.01).Immunohistochemistry demonstrated a brown round α-synuclein immunoactive mass in cytoplasm;cell immunofluorescence showed a strong greenα-synuclein immunoactive aggregation in cytoplasm.Viable apoptotic cells,non-viable apoptotic cells and necrosis cells were observed gradually as cell concentration increased and time prolonged.Conclusion Rotenone should be toxic to dopaminergic neurons,causing inclusion of α-synuclein aggregation and inducing apoptosis.The malmetabolism of α-synuclein and apoptosis induced by rotenone might play an important role in the pathogenisis of Parkinson's disease.