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目的:利用原子力显微镜等方法,明确α-突触核蛋白(α-synuclein,α-syn)过表达对线粒体的影响。方法:将腺相关病毒(AAV)载体介导表达的α-syn(AAV/α-syn)病毒包装颗粒,给大鼠脑内黑质区定位注射,建立α-syn过表达的大鼠模型,并以AAV介导表达的报告基因LacZ(AAV/LacZ)为动物对照组。提取大鼠黑质脑组织的线粒体,并通过JC-1染色检测线粒体膜电势(ΔΨ)变化和Western blot检测线粒体中α-syn蛋白量的变化;采用原子力显微镜观测线粒体表面结构的变化。结果:实验组大鼠黑质脑组织过表达α-syn基因,第16周时,Western blot显示线粒体中α-syn蛋白量增加约2倍;JC-1染色发现ΔΨ下降;原子力显微镜观测可见线粒体体积增大,线粒体膜表面出现较多孔道样改变。结论:大鼠黑质脑组织过表达α-syn基因,第16周时,可导致α-syn蛋白在线粒体积聚;并可引起线粒体增大、膜形成孔道样改变、ΔΨ下降。
OBJECTIVE: To determine the effects of α-synuclein (α-syn) overexpression on mitochondria by means of atomic force microscopy. Methods: The α-syn (AAV / α-syn) virus-encapsulated particles mediated by adeno-associated virus (AAV) vector were inserted into the substantia nigra region of rat brain to establish a rat model of α-syn overexpression. The reporter gene LacZ (AAV / LacZ) mediated by AAV was used as the animal control group. Mitochondria were extracted from the substantia nigra of rat brain and the changes of mitochondrial membrane potential (ΔΨ) were detected by JC-1 staining. The changes of α-syn protein in mitochondria were detected by Western blot. The changes of mitochondrial surface structure were observed by atomic force microscopy. Results: The α-syn gene was overexpressed in the substantia nigra of brain tissue of rats in experimental group. At week 16, the amount of α-syn protein in mitochondria was increased about 2-fold by Western blot. The ΔΨ was decreased by JC-1 staining. As the volume increases, more channel-like changes appear on the surface of mitochondrial membrane. CONCLUSIONS: α-syn gene is overexpressed in the substantia nigra of brain tissue at week 16, which leads to the accumulation of α-syn protein in mitochondria. It also leads to the increase of mitochondria, the change of pore-like membrane and the decrease of ΔΨ.