神经生物学相关论文摘要.docx

神经生物学相关论文摘要 在MN9D细胞α-synuclein N、NAC、C端结构域与线粒体关系的研究 张韬 硕士2009 帕金森病（PD）是最常见的神经退行性疾病之一，a-突触核蛋白（a-synuclein）及线粒体与PD发病密切相关，但作用机制尚不明确。a-synuclein是PD病理特征Lewy体的主要成分, a-synuclein基因突变可导致家族性PD，大多数PD患者存在线粒体功能障碍。本课题前期工作证实a-synuclein可位于线粒体，其在大鼠体内长期过表达可引起神经元线粒体结构变化，导致线粒体退行性变。本实验的目的是研究a-synuclein各结构域与线粒体结构功能的关系。我们成功构建了a-synuclein 基因的各结构域：pLNCX2/N，pLNCX2/NAC及pLNCX2/C的逆转录病毒的表达质粒，通过病毒包装获得了可表达a-synuclein各结构域的MN9D细胞。利用免疫细胞化学方法，通过激光扫描共焦显微镜发现a-synuclein/N端与线粒体存在共定位关系；a-synuclein/NAC主要在核内聚集表达；a-synuclein/C端在胞浆和胞核内均有表达。JC1染色流式细胞仪检测显示，过表达a-synuclein/N实验组细胞线粒体膜电位降低，同时CCK8检测结果显示该组细胞活力较正常组及其它处理组明显下降。凋亡检测显示，过表达a-synuclein/N可引起AIF总量增加及核转位，刺激细胞色素C释放，吖啶橙检测发现引起核DNA断裂以及线粒体形态发生变化。Annexin V/PI染色的直接证据表明a-synuclein/N参与凋亡并最终导致细胞死亡。电镜形态学观察发现a-synuclein/N可导致线粒体形态发生明显改变。因此我们认为a-synuclein的 N端可定位于线粒体，具有细胞毒性作用，并参与调节线粒体功能；a-synuclein/NAC虽具有疏水特性但却能穿过核膜，聚集在核仁周围，发挥着核内调控作用；a-synuclein/C端定位于胞浆和核内可能参与多种细胞功能且不排除其参与细胞保护作用。 Mitochondrial dysfunction is implicated in the pathophysiology of Parkinson’s disease characterized by formation of intraneuronal inclusions, Lewy bodies and progressive degeneration of the nigrostriatal dopamine system. Recently, pathophysiological functions of a-synuclein, a major constituent of Lewy bodies, have been shown in its interaction with mitochondria. Our present study is to identify which domain of a-synuclein is to be the key in affecting mitochondrial function, and how the function is to be impaired by alteration of a-synuclein. For this purpose, an N-terminal (amino acids 1-65), NAC (61-95), and C-terminal (91-140) fragments of a-synuclein were generated, and retroviral expression system was employed for transfection. Confocal microscopy, flow cytometry, Western blot analysis and electron microscope techniques were employed to evaluate co-localization of target protein and mitochondrion, mitochondrial membrane potential, mitochondrial modality, and direct interaction of interested proteins in MN9D cells. The results demonstrated that only the N-terminal fragment of a-synuclein was lo