【作者】 洪岩；

【导师】 朴丰源；

【作者基本信息】 大连医科大学 ， 劳动卫生与环境卫生学， 2009， 硕士

【摘要】 目的:研究亚慢性砷暴露对小鼠脑组织线粒体复合体ⅡSdha的表达影响,为探讨砷的神经毒性机制提供分子毒理学依据。方法:SPF级小鼠50只,雌雄各半,体重26.3-30.9 g。按体重将小鼠随机分为5组:1 ppm三氧化二砷(As2O3)染毒组;4 ppm As2O3染毒组;牛磺酸抗氧化干预组(4 ppm As2O3+150 mg/L牛磺酸)和维生素C抗氧化干预组(4 ppm As2O3+45 mg/L维生素C);蒸馏水对照组。动物自然饮水和饮食,室温18～25℃。通过饮用含不同浓度As2O3蒸馏水的方式使小鼠暴露于砷。保护剂是以灌胃方式投给,每星期2次。连续染毒60天,颈椎脱臼法处死小鼠。应用免疫组织化学8-羟基脱氧鸟嘌呤(8-OH-dG)实验检测小鼠脑组织氧化性损伤。应用电镜技术观察砷暴露小鼠脑组织线粒体的超微结构变化。应用基因芯片技术筛选染砷小鼠脑组织线粒体呼吸链复合体差异表达的相关基因。用western blot方法验证线粒体复合体ⅡSdha的蛋白表达。通过比色法观察砷对SDH的酶活性影响。采用SPSS10.0统计软件,用单因素方差分析(ANOVA),比较各染砷组与对照组间的统计学差异,以P <0.05表示统计学差异显著。结果:与对照组比较,4 ppm染砷组小鼠脑组织出现明显的8-OH-dG表达,而对照组未见8-OH-dG表达。电镜结果显示,对照组小鼠脑组织线粒体双层膜结构完整,嵴清晰可见。染砷组小鼠脑组织线粒体双层膜结构完整,线粒体肿胀,部分嵴消失或断裂,线粒体出现空泡变。基因芯片筛选结果显示:染砷组小鼠脑组织线粒体呼吸链复合体差异表达的相关基因共11条,其中表达下调的基因10条,分别为NADH-泛醌还原酶基因(Ndufab1, Ndufb12, and Ndufb2),琥珀酸-泛醌还原酶基因(Sdha),泛醌-细胞色素C还原酶基因(Uqcr),细胞色素C氧化酶基因(Cox6a2, Cox17),ATP合酶基因(Atp5a1, Atp5g1, Atpif1)。表达上调的基因为NADH-泛醌还原酶基因(Ndufs4)。小鼠脑组织线粒体复合体ⅡSdha的western blot结果显示:Sdha蛋白表达量随着染砷剂量的增加其蛋白表达量明显减少,尤其在4 ppm染砷组最为明显。在干预组(牛磺酸和维生素C),线粒体复合体ⅡSdha蛋白表达量比4 ppm染砷组明显增多。线粒体复合体ⅡSdha蛋白表达量随着染砷天数的增多而降低。染砷组小鼠脑组织SDH酶活性低于对照组,且随染砷浓度的增高而降低,尤其4 ppm染砷组SDH酶活性降低最为明显(P <0.05)。在牛磺酸和维生素C干预组,小鼠脑组织线粒体SDH酶活性显著高于4 ppm染砷组但低于对照组,差别有统计学意义(P <0.05)。结论:线粒体是砷毒性作用的亚细胞器,线粒体复合体Ⅱ可能是砷毒性作用的靶部位,Sdha可能是砷毒性作用的靶点。牛磺酸和维生素C干预剂对砷诱导的Sdha基因的毒性具有一定的干预作用。更多还原

【Abstract】 Objective:To examine the influence of subchronic exposure arsenic (As) on expression of Sdha protein in the brain of mice so as to provide the evidence of molecular target for exploring mechanism of As neurotoxicity.Methods: 50 mice were divided into 5 groups with 10 for each, Group 1 received drinking distilled water alone (control). Group 2 and 3 received 1 or 4 mg/L arsenic trioxide. Group 4 and 5 received both 4 mg/L arsenic trioxide and 150 mg/kg taurine or 45 mg/kg Vit C (as interfering groups), respectively. Arsenic trioxide was given through drinking water for 60 days and taurine or Vit C was poured into stomach twice a week. On the 60th day after As exposure, mice were decapitated and their brains were removed. 8-OH-dG expressions were examined by immunohistochemistry. Changes of mitochondrial ultrastructures were examined in brain subchronically exposed to arsenic by the electron microscope. Expression profiles of mitochondrial respiratory chain -related genes were examined in the brain of mice exposed to As by GeneChip. Level of Sdha protein expression and SDH activity were examined by western blot and colorimetry.Date were presented as mean±standard deviation(SD). All data were analyzed with SPSS10.0 for windows. Difference in mean values between groups was tested with the one-way ANOVA. P values less than 0.05 were considered significant.Results: Compared to control group, Intensive 8-OH-dG immunoreactivity was found in 4 ppm group. Changes of mitochondrial ultrastructures in the control group showed a double membrane of the wall and membranes of cristae were well arranged. In the mice given 4 ppm arsenic trioxide, morphological changes including swelling and vacuolus alteration were shown in mitochondria, but the double membranes of mitochondria remained in good shape. Among 11genes selected by gene chip, 10 genes were downregulated, namely, NADH dehydrogenase genes (Ndufab1, Ndufb12, and Ndufb2), succinate dehydrogenase gene (Sdha), ubiquinol-cytochrome c oxidoreductase gene (Uqcr), cytochrome oxidase genes (Cox6a2, Cox17) and ATP Synthase genes (Atp5a1, Atp5g1, Atpif1). NADH dehydrogenase gene (Ndufs4) was upregulated. The activity of SDH and level of Sdha protein expression in brain mitochondria decreased in the groups exposed to As. Especially, the SDH activity and level of Sdha protein expression were significantly lower in the group received 4 ppm As2O3 than those in controls (P <0.05). However, the activity of SDH and level of Sdha protein expression was significantly higher in interfering groups than those in the group received 4 ppm As2O3 (P <0.05). In this 4 ppm group, the SDH activity and level of Sdha protein expression were significantly lower on the 30th or 60th day after exposure than those on 0 day (P <0.05).Conclusion: Mitochondria may be a subcellular organelle of the arsenic neurotoxicity. mitochondria complexⅡmay be the most possible target site of the arsenic neurotoxicity. Sdha may be the most possible target of the arsenic neuron- toxicity. The toxic effect of As on Sdha was mitigated by the coadministration of taurine or Vit C.更多还原