三氧化二砷对小鼠卵母细胞线粒体DNA氧化损伤作用及其机制

发布时间：2018-03-30 16:19

本文选题：三氧化二砷　切入点：活性氧　出处：《中国药理学与毒理学杂志》2015年05期

【摘要】：目的研究三氧化二砷(As2O3)对小鼠卵母细胞线粒体DNA(mt DNA)的氧化损伤作用及其作用机制。方法 1体外实验挤压正常小鼠卵巢获取卵母细胞,培养成熟后,分为As2O31和2μmol·L-1单独处理组、N-乙酰半胱氨酸(NAC)5 mmol·L-1处理组、2,2,6,6-四甲基哌啶-1-氧基(Tempo)1 mmol·L-1处理组、As2O31或2μmol·L-1+NAC 5 mmol·L-1共处理组、As2O31或2μmol·L-1+Tempo 1 mmol·L-1共处理组,培养20 h后,收集成熟卵母细胞进行后续实验指标测定。2体内实验雌性昆明小鼠ip给药,分为As2O31和2 mg·kg-1染毒组、As2O31或2 mg·kg-1+NAC 200 mg·kg-1组,每日1次,连续60 d,椎脱臼处死取卵母细胞。2′,7′-二氯荧光黄双乙酸盐荧光染色检测细胞内活性氧(ROS)水平;8-羟基脱氧鸟苷酶(8-OHd G)联免疫反应检测提取mt DNA中8-OHd G含量;Western蛋白印迹法检测DNA聚合酶γ(Polγ)和线粒体转录因子A(mt TFA)的蛋白表达;β-半乳糖苷酶(β-Gal)报告基因检测试剂盒检测溶酶体活力。结果 1体外实验As2O3单独处理组小鼠卵母细胞中ROS和8-OHd G含量升高,Polγ和mt TFA蛋白表达水平降低(P0.05);而As2O3与NAC或Tempo共处理组卵母细胞中ROS含量减少,8-OHd G水平降低,Polγ和mt TFA蛋白表达水平显著上调(P0.05)。2体内实验As2O3单独处理组小鼠卵母细胞中ROS和8-OHd G含量升高,Polγ和mt TFA蛋白表达水平降低(P0.05);As2O3与NAC共处理组小鼠卵母细胞中ROS含量减少,8-OHd G水平降低,Polγ和mt TFA蛋白表达水平提高(P0.05)。As2O3单独处理组体外培养的卵母细胞,β-Gal活力显著提高(P0.05),而As2O3与NAC或Tempo共处理组β-Gal活力显著下降(P0.05)。体内实验各处理组β-Gal活力无显著差异。结论砷暴露可诱导小鼠卵母细胞大量生成ROS,抑制线粒体基因组修复与复制相关因子Polγ及mt TFA的表达,同时改变溶酶体活力,最终诱发mt DNA氧化损伤。
[Abstract]:Objective to study the oxidative damage of arsenic trioxide (as _ 2O _ 3) on mouse oocyte mitochondrial DNA(mt DNA and its mechanism. It was divided into two groups: As2O31 and 2 渭 mol L ~ (-1) alone treated with N- acetylcysteine and 5 mmol L ~ (-1). After 20 h culture, as _ 2O _ 31 or 2 渭 mol ~ (-1) NAC _ (5) mmol L ~ (-1) co-treated with as _ 2O _ 31 or 2 渭 mol ~ (-1) Tempo _ (1) mmol L ~ (-1) were co-treated with as _ 2O _ 31 or 2 渭 mol ~ (-1) NAC _ 5 mmol L ~ (-1). The adult oocytes were collected for further study. 2 female Kunming mice were given IP administration in vivo. The mice were divided into two groups: As2O31 and 2 mg kg-1 exposed to As2O31 or 2 mg kg-1 NAC 200mg kg-1, once a day. For 60 days, the oocytes were killed by vertebrae dislocation. The 8-OHd G content in mt DNA was detected by Western blot with fluorescence staining method of reactive oxygen species (Ros) and 8-OHd GG from mt DNA by fluorescence staining with fluorescein yellow diacetate. The protein expression of DNA polymerase 纬 -Pol 纬) and mitochondrial transcription factor A(mt TFAwere detected, and the lysosomal activity of 尾 -galactosidase (尾 -galactosidase) reporter gene assay kit was detected. Results 1in vitro As2O3 alone treated mouse oocytes with ROS and 尾 -galactosidase were detected for lysosomal activity. The expression of Pol 纬 and Mt TFA protein decreased in the oocytes treated with As2O3 and NAC or Tempo, while the level of ROS decreased in the oocytes treated with NAC or Tempo. The expression levels of Pol 纬 and Mt TFA in the oocytes treated with As2O3 or NAC or Tempo were significantly up-regulated in the P0.05n.2 group treated with As2O3 alone. Expression of ROS and 8-OHd G in mouse oocytes increased Pol 纬 and Mt TFA protein expression in mouse oocytes decreased ROS content in mouse oocytes treated with P0.05As2O3 and NAC, decreased 8-OHdG level, increased Pol 纬 and mt TFA protein expression level in mice oocytes treated with P0.05U 路As2O3 alone. The 尾 -Gal activity of oocytes cultured in vitro increased significantly, while the 尾 -Gal activity of As2O3 co-treated with NAC or Tempo decreased significantly. There was no significant difference in 尾 -Gal activity among the experimental groups in vivo. Conclusion arsenic exposure can induce the formation of large amounts of mouse oocytes. Ross. inhibit the expression of Pol 纬 and Mt TFA related to mitochondrial genome repair and replication. At the same time, the lysosome activity was changed and the oxidative damage of Mt DNA was induced.
【作者单位】：兰州大学生命科学学院甘肃省环境生物监测与修复重点实验室;
【基金】：国家自然科学基金(31300437) 甘肃省自然科学基金(1308RJZA139)~~
【分类号】：R114

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