α-突触核蛋白各结构域与MN9D细胞线粒体的关系

DIFFERENT DOMAINS OF α-SYNUCLEIN INVOLVED IN MITOCHONDRIAL OF MN9D CELLS

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。

Objective To investigate the interaction of different domains of α-synuclein gene with mitochondrial by overexpressing N-terminal, NAC and C-terminal of α-synuclein in MN9D cell. Methods N-terminal of α-synuclein/1-65 （amino acid, aa）, NAC of α-synuclein/61-95aa and C-terminal of α-synueIein/96-140aa were generated by PCR amplification and these cDNA fragments were then subcloned into retroviral pLNCX2 vectors. The reconstructed plasmids were transfected into PT67 packaging cell line by Lipofectin and viral particles were selected. After using the viral particle to infect the MN9D cells, the cell viability was evaluated by Cell Counting Kit-8 assay （CCK-8）. Gene expressing level was detected by real Time RT-PCR. The different domains of α-synuclein distribution and α-localization of target protein with mitochondrial were measured by immunofluorescence and the mitochondrial membrane potential was determined by flow eytometry. Results N-terminal of α- synuclein was found to be located together with mitochondrial and to make the membrane potential of mitochondrial decline, α- synuclein/NAC expressed mainly in nuclear while α-synuclein/C was chiefly found in cytoplasm. Conclusion N-terminal of α- synuclein may interact with mitochondrial and interfere its function by depolarizing its membrane potential, α-synuclein/NAC appears aggregating around the chromatospherite even though it has strong lyohobic characteristics, α-synuclein/C may perform its action mainly in cytoplasm.