【作者】 陈中华；

【导师】 张红雨； 张秀珍；

【作者基本信息】 山东理工大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 随着环境污染的日益严重,到达地球表面的紫外线日益增多,紫外线辐射会导致皮肤细胞的活性氧自由基增多,促进皮肤光老化、癌变、白内障、免疫抑制等一系列疾病的发生,因此寻找高效的抗氧化剂是预防和治疗紫外损伤的重要手段。本研究以NIH3T3细胞为实验材料,建立了紫外辐射对NIH3T3细胞的损伤模型,采用MTT方法和激光扫描共聚焦显微镜技术研究不同浓度的姜黄素、槲皮素、石杉碱甲对NIH3T3细胞的紫外损伤保护作用。结果表明,姜黄素预处理不但可以降低紫外损伤造成的细胞死亡率(p＜0.05)、减少细胞内活性氧的堆积(p＜0.05),而且对细胞线粒体膜电位具有保护作用(p＜0.01);此外,姜黄素对紫外损伤细胞的保护作用与浓度呈正相关。实验结果得出姜黄素可以清除细胞内的活性氧自由基,维持细胞的线粒体膜电位,提高紫外辐照细胞的存活率。通过不同浓度的槲皮素的预处理,紫外损伤后的细胞内活性氧自由基含量明显降低(p＜0.05),阻止了细胞膜电位下降(p＜0.05),提高了细胞存活率(p＜0.05);且都呈现出较强的槲皮素浓度依赖性。结果表明紫外损伤可诱导细胞内产生大量的活性氧自由基,使细胞线粒体膜电位下降,降低细胞的存活率;槲皮素预处理,可以清除细胞内的活性氧自由基,维持细胞的线粒体膜电位,抑制细胞凋亡。经过不同浓度的石杉碱甲的预处理后的NIH3T3细胞,经紫外损伤后,其细胞死亡率降低(p＜0.05)、细胞内活性氧的含量减少(p＜0.01),而且起到了对细胞线粒体膜电位的保护作用(p＜0.01);同时,石杉碱甲预处理后的NIH3T3细胞的细胞核形态相对保持完整,细胞凋亡过程中的关键蛋白caspase-3的表达量下降(p＜0.01),bcl-2的表达量增加(p＜0.01),实验结果表明,石杉碱甲可以有效的清除NIH3T3细胞内的活性氧自由基,保护细胞线粒体的膜电位,通过调控蛋白表达,抑制由紫外辐射诱导的NIH3T3细胞凋亡,提高细胞存活率。更多还原

【Abstract】 Studies have shown that chronic repeated exposures to sunlight are epidemiologically shown to be the main cause of skin cancers, skin aging, erythema, photoaging, immune suppression. At the cellular level, UV radiation causes significant oxidative stress due to generation of reactive oxygen species such as singlet oxygen, hydroxyl radical, superoxide anion, and hydrogen peroxide, then, our study set up a damage model with NIH3T3 cells by exposure it to UV radiation; investigate the antioxidative effects of curcumin, quercetin and huperzine A against the damage produced by exposing to UV in the cells.For curcumin and quercetin, the vitality of cells was determined by MTT assay; the membrane potential (MMP) across the inter membrane of the mitochondrial and the ROS level in the cells were evaluated by the laser scanning confocal microscopy. The study find out that exposure cells to UV caused a marked increase in ROS, and a decrease in mitochondrial membrane potential (MMP). Pre-incubation of NIH3T3 cells with curcumin and quercetin decrease the intracellular accumulation of ROS, and these two antioxidants caused a marked increase of MMP in 3T3 cells, they can protect cells against UV irradiation, increase the cell viabilty.For huperzine A, we use MTT assay to examine the vitality of cells and LSCM to determine the MMP, intracellular ROS level, cell karyotype, caspase-3 protein expression and bcl-2 expression. We find out that huperzine A can decrease the UV induced intracellular accumulation of ROS, increase the MMP of cells, keep the cell karyotype integrity; inhibit the UV induced apoptosis by regulate protein caspase-3 and bcl-2 expression, increase the viability of cells.更多还原