【作者】 赵杰；

【导师】 曹学兵； 孙圣刚；

【作者基本信息】 华中科技大学 ， 神经病学， 2008， 硕士

【摘要】 目的探讨溶酶体和蛋白酶体功能障碍在帕金森病发病中的作用机制。方法用神经生长因子(Nerve growth factor, NGF)诱导分化PC12细胞作为研究多巴胺能神经元的细胞载体,分别及联合应用溶酶体抑制剂和蛋白酶体抑制剂lactacystin、E64 (Trans-Epoxysuccinyl -L-leucylam)处理神经元样分化的PC12细胞,四甲基偶氮唑盐(MTT)法检测细胞活性及代谢状态、流式细胞术检测细胞凋亡率,免疫荧光双标方法观察PC12细胞内硫黄素S、α-synuclein蛋白阳性聚集包涵体的形成;Hoechst33258观察α-synuclein蛋白、硫黄素S双标阳性细胞的凋亡。结果应用上述抑制剂后细胞活性下降(呈浓度依赖性)而细胞凋亡率上升;药物处理后出现α-synuclein蛋白聚集和包涵体形成的PC12细胞百分比为: E64组7.94±0.97%,lactacystin组20.33±2.4%,合用组36.77±3.5%,与对照组及各组之间相比差异均有显著性(P<0.05);α-synuclein蛋白阳性包涵体细胞存在明显凋亡(17.29±1.54%)。结论溶酶体和蛋白酶体功能障碍通过诱导多巴胺神经元细胞内α-synuclein蛋白异常聚集可能在PD发病机制中发挥重要作用。目的探讨α-synuclein蛋白细胞内溶酶体途径降解方式及其聚集机制。方法用神经生长因子NGF诱导分化PC12细胞作为研究多巴胺能神经元的细胞载体,应用鱼藤酮处理PC12细胞建立α-synuclein蛋白细胞模型。用LAMP2抗体标记溶酶体膜,用免疫荧光双标明确α-synuclein蛋白和LAMP2之间的共定位。使用溶酶体途径降解抑制剂E64 ,3-MA,BafA1处理神经元样分化的PC12细胞,应用免疫荧光双标方法观察PC12细胞内硫黄素S、α-synuclein蛋白阳性聚集包涵体形成情况。比较各组的差异。结果α-synuclein蛋白在溶酶体中出现,用E64处理鱼藤酮预处理过的PC12细胞后α-synuclein蛋白聚集且较多包涵体形成15.36±0.85%,与对照组、3-MA组和BafA1组相比有统计学意义(P<0.05);3-MA,BafA1处理鱼藤酮预处理过的PC12细胞后α-synuclein蛋白聚集较少且仅有少量包涵体形成(7.65±0.46%、8.72±0.39%),与对照组相比无统计学意义(P>0.05)。结论溶酶体分子伴侣介导自噬途径可能在α-synuclein蛋白降解、聚集和多巴胺神经元死亡过程中发挥重要作用。更多还原

【Abstract】 Objective To investigate the effect and mechanism of the lysosome dysfunction and the proteasome dysfunction in Parkinson disease (PD).Methods PC12 cells as dopaminergic neurons, differentiated by nerve growth factor, were treated by lysosome inhibitor E64 and proteasome inhibitor lactacystin. Cell viability was measured by MTT assay; Flow cytometry was used to detect cell apoptosis; Protein aggregation was detected by the double labeling imunofluorescence staining of Thioflavine S andα-synuclein; the apoptosis of the double labeling cells was examined by Hoechst33258 staining.Results The cell viability declined in a concentration-dependent manner while the apoptosis ratio increased when treated with the lysosome inhibitor E64, the proteasome inhibitor lactacystin and lactacystin+E64 in turn; E64 and lactacystin inducedα-synuclein protein aggregate and lead to the formation of eosinophilic intracytoplasmic inclusions, which was immunoreactive toα-synuclein and Thioflavine S. with 7.94±0.97% and 20.33±2.4%; Theα-synuclein protein aggregation became more significant(36.77±3.5%)when the two inhibitors were used by combination(P<0.05); Some inclusion-harboring cells revealed positive Hoechst33258 staining(17.29±1.54%).Conclusions The lysosome dysfunction and the proteasome dysfunction play an important role in the metabolism ofα-synuclein, inclusions formation and dopaminergic neuronal death. Objective To investigate the pathway and mechanism ofα-synuclein in lysosomal degradation pathway.Methods PC12 cells as dopaminergic neurons, differentiated by nerve growth factor. PC12 cells were treated by Rotenone to makeα-synuclein model. Then PC12 cells were treated by different lysosomal inhibitors E64, 3-MA, BafA1. Protein aggregation was detected by the double labeling of Thioflavine S andα-synuclein for imunofluorescence staining.Results E64 inducedα-synuclein protein aggregate and lead to the formation of eosinophilic intracytoplasmic inclusions, which was immunoreactive toα-synuclein and Thioflavine S. with 15.36±0.85% (P<0.05). Theα-synuclein protein aggregation was not significant (7.65±0.46%、8.72±0.39%)when the macroautophagy inhibitors 3-MA and microautophgy inhibitors BafA1 were used (P>0.05).Conclusions The chaperone—mediated autophagy pathway play an important role in the metabolism ofα-synuclein, inclusions formation and dopaminergic neuronal death.更多还原