目的探讨α-突触核蛋白（α-synuclein）各结构域与MN9D细胞线粒体的关系。方法用PCR方法获得α-synuclein/1~65（N），α-synuclein/61~95（A）及α-synuclein/96~140（C）基因片段，克隆入真核表达质粒pLNCX2，经测序正确后以脂质体转染PT-67细胞，挑取单克隆并扩增，收集上清感染MN9D细胞，分别用Real Time PCR检测细胞基因表达水平，免疫组织化学染色检测蛋白表达，激光扫描共焦显微镜检测蛋白与线粒体共定位，流式细胞术检测细胞状态及线粒体膜电位状态。结果成功构建了pLNCX2/N、pLNCX2/NAC及pLNCX2/C基因片段的重组真核表达质粒，获得了可稳定表达α-synuclein各基因片段的MN9D细胞株。通过激光扫描共焦显微镜观察，可见α-synuclein/N端与线粒体存在共定位关系；α-synuclein/NAC主要在核内聚集表达；α-synuclein/C端在胞质和胞核内均有表达。JC1染色流式细胞术检测显示，过表达α-synuclein/N实验组细胞线粒体膜电位降低。结论α-synuclein的N端可能定位于线粒体，并参与调节线粒体功能；α-synuclein/NAC虽具有疏水性但却能穿过核膜，聚集在核仁周围；α-synuclein的C端定位于胞质和核内可能参与多种细胞功能。
This study was designed to investigate the effects of α-synuclein on toxicity induced by long-term exposure to relatively low concentrations of rotenone.Compared with the control groups,the inhibition of cell viability which overexpressed α-synuclein(SH-SY5Y-Syn) improved after 1 and 2 weeks of rotenone treatment.The complex I activity was greater and the mitochondrial membrane swelling intensity was reduced after 1 and 2 weeks of treatment,which indicated that α-synuclein,at least in part,resists the rotenone-induced oxidative stress.The results indicate that α-synuclein has a dual effect on toxicity of rotenone according to exposure time in human SH-SY5Y cells.
LU LingLing,GU Li,LIANG Yuan,SUN XiaoHong,DUAN ChunLi & YANG Hui Beijing Institute for Neuroscience,Capital Medical University,The Beijing Center of Neural Regeneration and Repairingr,Key Laboratory for Neurodegenerative Disease of the Ministry of Education,Beijing 100069,China
