【作者】 刘曼；

【导师】 姬汴生；

【作者基本信息】 河南大学 ， 药理学， 2011， 硕士

【摘要】 目的:探讨多胺类化合物（polyamine analogues ,WJ01）对缺氧缺糖,天然多胺（putrescine,Put）和谷氨酸（glutamate,Glu）诱导的PC12细胞（大鼠嗜铬细胞瘤细胞,pheochromocytoma cell）损伤的保护作用及其机制。方法:运用不同的方法造成PC12细胞损伤模型,通过倒置显微镜观察细胞的形态,MTT法检测PC12细胞存活率,研究多胺类化合物的细胞保护作用;采用吖啶橙（Acridine Orange,AO）和溴化乙锭（Ethidium bromide,EB）双染色法观察细胞凋亡形态和特征;用DCFH-DA检测细胞内ROS的水平,罗丹明123（Rh123）染色法测定细胞线粒体膜电位的变化;用钙离子敏感的荧光探针Fura-2/AM检测细胞内游离Ca²⁺浓度;从而进一步研究多胺类化合物的细胞保护作用的可能性机制。结果:1.经缺糖缺氧培养后,细胞存活率显著降低,多胺化合物能明显改善受损细胞形态,显著提高MTT值及细胞存活率,对连二亚硫酸钠造成PC12细胞缺氧损伤表现出保护作用。2.天然多胺可诱导PC12细胞损伤,提高细胞内的ROS水平,增加细胞内Ca²⁺内流,降低线粒体膜电位,多胺类化合物可改善细胞形态,提高细胞存活率,降低ROS水平,减少细胞内Ca²⁺内流,抑制线粒体膜降低,减少细胞凋亡。3.经谷氨酸毒性损伤,多胺类化合物可改善细胞形态,提高细胞存活率,减少细胞内Ca²⁺内流,降低ROS水平,抑制线粒体膜降低,减少细胞凋亡。结论:通过离体实验证明多胺类化合物对缺氧缺糖、天然多胺及谷氨酸诱导的PC12细胞损伤有保护作用,为进一步揭示其对脑缺血损伤的作用机制提供依据。更多还原

【Abstract】 Objective: To investigate the protective effect of polyamine analogues （WJ01） on the oxygen-glucose deprivation, glutamate （Glu） and putrescine （Put） induced cytotoxicity in rat pheochromocytoma cells （PC12）.Methods: Different models were used to induce the cytotoxicity in PC12 cells, the morphology of PC12 cells was observed by microscope and the cell damage was measured by MTT assay. The reactive oxygen species （ROS） were monitored combined with DCFH-DA by High-Content Screening Reader. DNA-binding dye Acridine Orange （AO） and Ethidium bromide （EB） were used to determine the morphological characteristic of apoptotic cells. Mitochondrial membrane potential （MMP） in PC12 cells were monitored by fluorospectrophotometer combining with Rh123. Fura-2/AM, a cell permeable fluorescent probe, was employed to detect intracellular Ca²⁺ concentration ([Ca²⁺]i).Results: 1. After treatment with 2 mM Na₂S₂O₄ in PC12 cells for 30 min, the cell viability decreased markedly, while WJ01 at the different concentrations （1μM, 5μM, 10μM） could elevate the cell viability obviously. 2. The results suggested that 12.5μM Put decreased the cell viability and the mitochondrial membrane potential, in contrast, Put increased ROS and intracellular [Ca²⁺]i. WJ01 significantly decreased ROS and intracellular [Ca²⁺]i, increased MMP in PC12 cells. 3. After the treatment with 50 mM Glu, the mitochondrial membrane potential significantly decreased while ROS and intracellular [Ca²⁺]i increased, and microscopic observation showed that PC12 cells exhibited morphological alterations such as cell shrinkage and membrane blabbing, the typical characteristics of apoptotic cell death, which was reduced with co-treatment of WJ01. Conclusions: The study shows that WJ01 at the different concentration has a protective effects against Na₂S₂O₄, Put and Glu-induced cytotoxicity and apoptosis in PC12 cells, which may represent the cellular mechanisms including scavenging ROS, increasing MMP, inhibiting the cell apoptosis and overload of Ca²⁺ in PC12 cells. WJ01 may represent a potential treatment strategy for attenuating ischemia injury in PC12 cells.更多还原