α-synuclein-EGFP在体外培养SH-SY5Y细胞中过表达对包涵体形成及线粒体结构改变的影响

为了观察 α-synuclein-p EGFP真核载体在 SH-SY5 Y细胞内表达状况及其对细胞的影响 ,本研究用 L ipofect AMINE法转染 SH-SY5 Y细胞 ,用荧光显微镜检测报告基因表达产物 EGFP,用荧光免疫细胞化学法检测α-synuclein蛋白表达以及用 H-E染色、光学显微镜观察转基因表达产物在细胞内的积聚 ,电镜观察转基因细胞超微结构形态学变化 ,rhodamine12 3染色、荧光显微镜检测线粒体跨膜电位变化。结果证明 ,该载体转染 SH-SY5 Y细胞后 ,在细胞内同时表达报告基因和目的基因 ;外源基因的表达产物可在细胞内积聚形成包涵体并引起线粒体结构和功能改变。本研究结果提示 ,本实验获得的细胞模型可模拟 Parkinson病发生过程中一些基本的神经病理学特点 ,可为进一步探讨 Parkinson病患者多巴胺能神经元变性缺失机制提供依据

THE INFLUENCE OF OVEREXPRESSION α-SYNUCLEIN-EGFP ON FORMATION OF INCLUSION AND MITOCHONDRIAL ULTRASTRUCTURE IN SH-SY5Y CELLS IN VITRO

To observe the overexpression of α-synuclein-EGFP in SH-SY5Y cultured cells and investigate its influence on cells, α-synuclein-pEGFP was transfected into SH-SY5Y cells with LipofectAMINE. The expression of α-synuclein-pEGFP was determined by EGFP fluorescence and anti-α-synuclein immunocytochemistry. The aggregates in the cultured cells were identified with HE staining and anti-α-synuclein immunocytochemistry. Ultrastructural alterations in the transfected cells were determined with electron microscopy. The mitochondrial membrane potential was detected with rhodamine 123 staining. α-synuclein-pEGFP could express both α-synuclein and reporter gene, EGFP, in SH-SY5Y cells. The α-synuclein and EGFP fusion protein could aggregate and lead to formation of the inclusions, abnormality of the ultrastructure of the mitochondria and decline of the mitochondrial membrane potential. The cells with α-synuclein-EGFP overexpression could reproduce some pathological features of dopaminergic neurons from the brain with Parkinson's disease. This work would be helpful for further study on the roles of α-synuclein in the pathogenesis of Parkinson's disease.