【作者】 高怡；

【导师】 裴秋玲；

【作者基本信息】 山西医科大学 ， 卫生毒理学， 2004， 硕士

【摘要】 目的 1．观察不同剂量砷染毒大鼠肝细胞膜转运蛋白(MRP2)表达水平的变化与砷代谢的关系 2．观察用BSO抑制大鼠谷胱甘肽(GSH)合成酶，对肝脏砷代谢的影响 方法 30只健康Wistar大鼠随机分为5组，每组6只，雌雄各半，灌胃染毒，受试物为亚砷酸钠(分析纯)，以生理盐水溶解。第1组为对照组，给予生理盐水；第2～4组为染砷组，分别给予4，10和20mg／kg体重的亚砷酸钠溶液，隔天染毒一次，2周后将动物处死；第5组为BSO干预组，在实验结束的前3天，每天大鼠预先腹腔注射2mmol／kg体重BSO溶液，4小时后再经口给予20mg／kg体重的亚砷酸钠溶液，其他处理同上。原子吸收分光光度法测定肝组织、胆汁和全血中的总砷含量。蛋白印迹法测定肝细胞膜上MRP2的含量改变。在电镜和光镜下观察形态学改变，同时测定血清谷丙转氨酶(ALT)，总胆红素，谷胱甘肽(GSH)，谷胱甘肽过氧化物酶(GSH-PX)，丙二醛(MDA)等指标，观察肝脏受损情况。 结果 随着染砷剂量的增加，胆汁和肝脏中砷含量逐渐增加，经方差检验差异均有统计学意义(P＜0.05)。两两比较发现，3个染毒组的血砷与对照组之间差异有统计学意义，而3个染毒组间差异无显著统计学意义；随染砷剂量增加，MRP2表达明显增加，且MRP2表达量与胆汁砷含量呈正相关关系；随着染砷剂量增加，血清ALT活性也随之增加，高剂量组胆红素含量明显高于对照组。与对照组相比，高剂量组肝GSH含量及GSH一PX活力显著降低，中、高剂量组MDA水平则显著升高;大鼠经BSO预处理后再给予20 mg/kg体重的亚砷酸钠溶液，发现胆汁砷含量明显高于对照组，而低于高剂量组;肝脏砷含量明显高于对照组和高剂量组;MPRZ表达量比高剂量组略有下降;与高剂量组和对照组相比，血清ALT活性明显增高，胆红素含量明显升高，GSH含量及GSH一PX活力显著降低，MDA水平则显著升高。光镜下观察染毒组肝组织可见少量散在的脂肪性变，肝细胞坏死以及小叶间胆管上皮细胞增生的表现。透射电镜观察可见，随染毒剂量的增加肝组织超微结构病变有所加重，具体表现为核形不规则，线粒体显著肿胀，山脊减少，部分靖断裂、消失，还可见胆小管微绒毛变得稀疏，微肿胀。结论1.亚砷酸钠致使肝脏功能受损，并且随着染砷剂量增加，肝脏损害不断加重。亚砷酸钠可以诱导MRPZ的表达，随染砷剂量增加，MR陀表达量增加。MRPZ在砷及其代谢产物的胆汁排泄过程中发挥了重要作用。 2.MRPZ转运砷及其代谢产物的作用与细胞内GSH水平相关，BsO通过抑制GSH合成，导致MRPZ一GSH共转运功能减弱，从而增加砷对肝脏的毒性作用.更多还原

【Abstract】 Objective To determine whether expression of MRP2 increased with increasing arsenic concentration and to observe effects of BSO, an inhibitor of GSH synthesis on hepatic arsenic metabolism in rats.Methods Thirty healthy Wistar rats were divided randomly into five groups and challenged with different concentrations of sodium arsenite : 0, 4, 10 and 20 mgAs/kgBW. High dose animals were also pretreated with BSO before arsenic administration. All animals were sacrificed 2 weeks after this treatment. Arsenic in liven blood and bile were detected by atomic absorption spectroscopy (AAS), and expression of MRP2 in the membrane of hepatocyte was determined by Western-blot analysis. By light microscopy and eletric microscopy, the morphological changes of liver were observed. Five biochemical indexes: Alanine aminotransferase (ALT) , total bilirubin in serum, glutathione (GSH), glutathione peroxidase (GSH-PX),and malonicacid (MDA) were selected to show the toxic effects on rats.Results The level of total arsenic in blood, bile and liver at all three different dose groups were higher than those in control groups. Significant difference can seen between two dose group in arsenic level of bile and liver, but there is no difference between two dose group in arsenic level of blood. Expression of MRP2 increased with increasing arsenic concentration. A clear tendency for apositive correlation between biliary arsenic concentration and MRP2 levels was found in liver. The activity of ALT increased as the dose increasing and showed a dose-effect manner. Compared with controls, total bilirubin in serum was increased in high-dose group. The contents of GSH and the activity of GSH-PX in high-dose group were higher than those in controls. The level of MDA in middle and high-dose groups increased markedly. Pretreatment with BSO decreased GSH level and increased lipid peroxidation. Biliary arsenic of rats pretreated with BSO was lower than that in rats of high-dose group and pretreatment with BSO increased arsenic content in tissues of rats. Expression of MRP2 pretreated with BSO was slightly decreasing. There was a little proliferation and necrosis in hepatocytes by LM. In bile canaliculi microvilli became swelling and sparse and the shape of nuclear showed irregular and some of mitochondrial cristae missed or broken by EM compared with control groups. Conclusion Sodium arsenite can induced expression of MRP2. The up-regulation of MRP2 proteins play an important role in the bile secretion of arsenite and its metabolies. Depletion of GSH reduced the function of MRP2-GSH cotransport system.更多还原