【作者】 徐玲；

【导师】 姬汴生；

【作者基本信息】 河南大学 ， 药理学， 2010， 硕士

【摘要】 目的：探讨6-氟基丁基苯酞(3-butyl-6-fluoro-1 (3H)-isobenzofuranone, F-NBP)对H2O2(hydrogen peroxide)介导的PC12细胞(大鼠嗜铬细胞瘤细胞,pheochromocytoma cell)损伤的保护作用及其可能机制。方法：运用不同浓度的连二亚硫酸钠(1,2,4,8,10 mM)对PC12细胞进行损伤,通过MTT法了解在不同时间里PC12细胞的损伤情况,确定出引起细胞损伤的最佳作用浓度和作用时间,并研究了6-氟基丁基苯酞和对照品丁基苯酞的保护作用。运用不同浓度的H2O2(100,300,500,700,1000μM )对PC12细胞进行损伤,通过MTT法了解在不同时间里PC12细胞的损伤情况,确定出引起细胞损伤的最佳作用浓度和作用时间,并研究了6-氟基丁基苯酞和对照品丁基苯酞的保护作用。同时采用GSH-PX, MDA等试剂盒检测H2O2对PC12细胞的氧化损伤,并研究6-氟基丁基苯酞的抗氧化作用及其机制,为进一步确定6-氟基丁基苯酞的抗氧化特性,使用DCF-DA检测细胞内ROS的水平情况。另外,对H2O2介导的PC12细胞凋亡进行了进一步的探讨,采用荧光倒置显微镜、活细胞高内涵成像系统检测凋亡细胞的形态,用活细胞高内涵分析系统对细胞线粒体膜电位的水平也进行了分析。结果：2 mM的连二亚硫酸钠处理PC12细胞24小时损伤效果最好,而浓度为5μM,10μM,50μM的6-氟基丁基苯酞可显著提高PC12细胞损伤抑制率,分别达到13%,43%,82%,且具有浓度依赖性。500μM的H2O2处理PC12细胞24小时损伤效果最好,而浓度为5μM,10μM,50μM的6-氟基丁基苯酞可显著提高PC12细胞损伤抑制率,分别达到20%,50%,70%,且具有浓度依赖性。H2O2可显著诱导PC12细胞氧化损伤,造成胞内GSH-PX降低和MDA的增加,并使细胞内ROS水平升高,触发氧化应激对细胞进行损伤,6-氟基丁基苯酞对上述的氧化损伤有抑制作用,对PC12细胞具有保护作用。500μM的H2O2接触细胞24小时后,通过荧光倒置显微镜、活细胞高内涵成像系统观察发现凋亡细胞显著增加,而预先给与6-氟基丁基苯酞作用2小时,可明显降低细胞凋亡。另外,通过测定荧光染料罗丹明123(Rh123)的荧光强度可以反映细胞线粒体膜电位的变化,H2O2能显著降低线粒体膜电位的水平,而6-氟基丁基苯酞则具有一定的保护作用。结论：1.2 mM的连二亚硫酸钠可介导PC12细胞缺糖缺氧损伤,诱导细胞凋亡,最终导致细胞死亡。一定浓度的6-氟基丁基苯酞预处理2小时可明显抑制连二亚硫酸钠诱导的细胞损伤和凋亡的作用。2.500μM的H2O2可介导PC12细胞损伤,触发氧化损伤,诱导细胞凋亡,最终导致细胞死亡。一定浓度的6-氟基丁基苯酞预处理2小时可明显抑制H202诱导的细胞损伤和凋亡的作用。3.6-氟基丁基苯酞的神经保护作用可能是通过清除细胞内ROS,抑制细胞凋亡来实现的。6-氟基丁基苯酞可能在脑缺血的防治中有潜在的应用价值。更多还原

【Abstract】 Objective:To investigate the protective effect of 3-butyl-6-fluoro-1(3H)-isobenzofuranone on the H2O2-induced cytotoxicity in rat pheochromocytoma cells (PC12) cells.Methods:Different dose of Na2S2O4 was used to induce the cytotoxicity in PC 12 cells in order to optimize dose by MTT assay. Different dose of H2O2 was used to induce the cytotoxicity in PC 12 cells in order to optimize dose by MTT assay. The enzyme activity of superoxide dismutase(SOD), glutathione peroxidase(GSH-PX) and the lipid peroxidation products malondialdehyde(MDA) in the cells were observed. DNA-binding dye Acridine Orange(AO) was used to determine the morphological characteristic of apoptotic cells. PI was used to a signature of cell membrane integrity and cell damage.Mitochondrial membrane potential in PC 12 cells were monitored by fluorospectrophotometer combining with Rh123.Results:1. After treatment with 2mM Na2S2O4 in PC 12 cells for 24 hours, the cell viability decreased markedly, while F-NBP at the different concentration (5μm,10μM,50μM) could elevate the cell viability obviously.2. After treatment with 500μM H2O2 in PC 12 cells for 24 hours, the cell viability decreased markedly, while F-NBP at the different concentration (5μM,10μM,50μM) could elevate the cell viability obviously.3. The results suggest that H2O2 decreasedGSH-PX and GSH-PX activity, in contrast, H2O2 increased MDA and ROS. F-NBP significantly decreased ROS and MDA, increased GSH-PX activity in PC 12 cells.4. After the treatment with 500μM H2O2, the mitochondrial membrane potential significantly decreased, microscopic observation showed that PC 12 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 F-NBP.Conclusions:1.2 mM Na2S2O4 could induce ischemia and apoptosis in PC 12 cells, which result in the cell death ultimately.2.500μM H2O2 could induce toxicity, oxidative damage and apoptosis in PC 12 cells, which result in the cell death ultimately.3. The study shows that F-NBP at the different concentration has a protective effects against H2O2-induced cytotoxicity and apoptosis in PC 12 cells, which may represent the cellular mechanisms including scavenging ROS, increasing GSH-PX activity. F-NBP may also represent a potential treatment strategy for ischemia.更多还原