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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 relationship between the α-synuclein domains and the mitochondria of MN9D cells. Methods The α-synuclein / 1-65 (N), α-synuclein / 61-95 (A) and α-synuclein / 96-140 (C) fragments were obtained by PCR and cloned into the eukaryotic expression plasmid pLNCX2. After being correct, PT-67 cells were transfected with liposomes. The single clones were picked and amplified. The supernatant was collected to infect MN9D cells. Real-time PCR was used to detect the gene expression of PTEN. The protein expression was detected by immunohistochemical staining. The co-localization of protein and mitochondria was detected by focal microscopy, and the cell status and mitochondrial membrane potential were detected by flow cytometry. Results The eukaryotic expression plasmids pLNCX2 / N, pLNCX2 / NAC and pLNCX2 / C were successfully constructed and the MN9D cell lines stably expressing α-synuclein gene were obtained. Α-synuclein / N-terminal co-localization with mitochondria was observed by laser scanning confocal microscopy; α-synuclein / NAC was mainly expressed in nucleus; α-synuclein / C-terminal was expressed both in cytoplasm and nucleus . JC1 staining showed that mitochondrial membrane potential of α-synuclein / N overexpression group decreased. Conclusion N-terminal α-synuclein may be located in the mitochondria and play a role in the regulation of mitochondrial function. Although α-synuclein / NAC is hydrophobic, it can cross the nuclear membrane and accumulate around the nucleolus. Quality and nuclear may be involved in a variety of cellular functions.