导　　师： 周玫

学科专业： 071010

授予学位： 硕士

作　　者： ;

机构地区： 中国人民解放军第一军医大学

摘　　要： 目的:该实验研究选用pc12细胞作为研究对象,将mnsod基因导入细胞中,使之在细 胞内稳定表达,然后观察h<,2>o<,2>对基因修饰后pc12细胞的细胞毒性作用变化,揭示mnsod在h<,2>o<,2>对pc12细胞的细胞毒中的作用,为以后研究神经系统疾病打下基础,为防治 脑氧应激损伤提供依据.结论:mnsod具有抑h<,2>o<,2>对pc12细胞的凋亡作用,也表明h<,2>o<,2>诱导pc12的凋亡作用可能涉及到损伤细胞的线粒体,导致氧化还原失衡继而诱发细 胞凋亡. Increasing evidence implicates oxidative stress in the neurodegenerative diseases,such as Alzheimer's disease/(AD/), Parkinson~ s diease, aging, ischemia and reperfusion. Hydrogen peroxide is an important precursor of highly reactive free radicals. Hydrogen peroxide can induced many cell lines apoptosis, one of the mechanism of which is by reactive oxygen species from mitochondria or via changing the mitochondrial membrane potential. Manganese superoxide dismutase /(MnSOD/) is strictly a mitochondrial enzyme located in the inner membrane and encoded by nuclear gene. It protects mitochondria from oxidative damage. So this study to explore that MnSOD modulates hydrogen peroxide induced apoptosis in the transformed PC12 cells. The sense and antisense human MnSOD cDNA under the transcriptional control of a human P -actin promoter were introduced into PC12 cells by Iipofectin transfection with recombinant plasmid containing neomycin selectable marker. MnSOD activity increased about two-fold in the cells transfected with the sense MnSOD cDNA, and decreased to about 33/% in the cells transfected with the antisense MnSOD cDNA. When PC12 cells were exposed to the low concentration /(50-lOOjtM/) of hydrogen peroxide, the direct consequence of oxidative damage is apoptosis. The cytotoxicity was assayed by MTT assay, flow cytometry was used to analyze DNA fragmentation quantitatively and Hoechst 33258 stain revealed the morphological changes of nucleus. But the cells transfected sense MnSOD gene can significantly prevent cells from the insult of hydrogen peroxide; on the other hand, the cells transfected with antisense MnSOD gene were more sensitive to hydrogen peroxide cytotoxicity than parent PCI2 cells. The expression of APP gene didn't alter in neither of the cells transfected with sense MnSOD eDNA or the cells transfected with antisense MnSOD cDNA. So the results show that the cells overexpressing MnSOD confer protection against H202-induced apoptosis on PC12 cells. And the results suggest the damage of mitochondria may be involved in the cytotoxicity of H202 on PC12 cells, which induces imbalance of cellular redox state and apoptosis.

关 键 词： 细胞凋亡 基因 细胞 过氧化氢

分 类 号： [Q5]

领　　域： [生物学]