关键词: α-突触核蛋白片段, 线粒体, 帕金森病, 流式细胞术, 免疫组织化学, MN9D细胞

Abstract: Objective To investigate the interaction of different domains of α-synuclein gene with mitochondrial by over-expressing 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-synuclein/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 co-localization of target protein with mitochondrial were measured by immunofluorescence and the mitochondrial membrane potential was determined by flow cytometry. 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

Key words: α-synuclein, Mitochondrial, Parkinson disease, Flow cytometry, Immunocytochemistry, MN9D cell