【作者】 陈曦；

【导师】 汤乃军；

【作者基本信息】 天津医科大学 ， 劳动卫生与环境卫生， 2009， 硕士

【摘要】 目的:研究低剂量二噁英慢性暴露对雌性大鼠卵巢和血清蛋白表达的影响,以进一步探讨二噁英生殖毒性和其它毒性机制,为寻找二噁英的蛋白生物标志物提供佐证。方法:对雌性SD-大鼠进行慢性染毒29周,分为三个染毒组和一个对照组(n=8/组,0ng/kg/d、20ng/kg/d、50ng/kg/d、125ng/kg/d四组),采用双向凝胶电泳(2-dimensional gel electrophoresis,2-DE)和基质辅助激光解吸飞行时间串联质谱(matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry,MALDI-TOF-MS/MS)对大鼠卵巢和血清进行蛋白组学研究,寻找差异蛋白点,并通过Western免疫印迹(Western Blot)技术,对所选择蛋白点进行反向验证;同时,对其肝脏进行组织病理学检测,应用酶联免疫吸附实验(enzyme linkedimmunosorbent assay,ELISA)测定血清中生殖激素水平(雌二醇、卵泡雌激素、黄体生成素),同时进行股骨密度的测定。结合大鼠体重资料,对实验结果进行统计学分析,进行讨论并得出结论。结果:卵巢中上调的蛋白包括:硒结合蛋白2(Selenium binding protein 2,SBP2),谷胱甘肽硫转移酶m类3型(glutathione S-transferase,mu type 3,GSTM3),Lrpap1蛋白,还原型辅酶Ⅱ(NADPH),Peptidylprolyl isomerase D(5414);下调的蛋白包括:抑制蛋白(Prohibitin,Phb)和N-乙基-马来酰胺敏感因子(N-ethylmaleimidesensitive factor,NSF),同时,在卵巢组织中得出Prohibitin反向验证结果。依据各种蛋白质的功能,在TCDD毒效应产生过程中发挥不同作用。TCDD染毒对于体重、卵巢脏器系数、生殖激素产生影响,蛋白表达的差异与血清雌二醇水平的改变相联系。提示TCDD在某种程度上扰乱了生殖激素的紊乱和相关调控蛋白的功能。虽然骨密度测定差别不具有统计学意义,但对照组的标准差较小。染毒组出现明显肝脏损伤,与多数研究结果一致,肝脏增生为二噁英所致肝脏损伤的重要特点,同时引起了肝脏绝对重量和相对重量的增加。血清中上调的蛋白包括补体C4(Complement component 4,C4),载脂蛋白A-IV前体(Preaploipoprotein A-IV)和低分子量的激肽原前体(LMW T-kininogen1 precursor);触珠蛋白(Haptoglobin,Hp)和补体C3(Complement component 3,C3)两种蛋白仅在染毒组大鼠的凝胶中有点被识别出。结论:二噁英慢性暴露对大鼠卵巢和血清蛋白表达产生了一定的影响,所鉴定的差异蛋白点有助于为进一步研究二噁英的生殖毒性和其他相关毒性提供科学依据;与激素水平、体重、脏器系数、组织形态的改变相联系,深入分析差异蛋白在TCDD毒作用过程中所发挥的作用和慢性低剂量暴露对雌性SD-大鼠的影响。更多还原

【Abstract】 Objectives 2,3,7,8-Tetrachlorodibenzo-p-dioxin（TCDD） is a ubiquitously distributed endocrine-disrupting chemical and reproductive toxicant.To elucidate its reproductive or endocrine toxicity and to investigate the differences of ovary and serum protein expression especially around the low-dose range with chronic exposure.Methods 32 female Sprague-Dawley rats were administered orally various concentrations(Ong·kg^-1·d^-1,20ng·kg^-1·d^-1,50ng·kg^-1·d^-1,125ng·kg^-1·d^-1) of TCDD for 29 weeks.Proteomic analysis of ovary and serum by two-dimensional gel electrophoresis and MALDI Tandem Mass Spectrometry showed distinct changes in the levels of several proteins that are relevant to TCDD toxicity.The hepatic histopathology was also performed after the sacrifice.Results Serum estradiol levels of TCDD-treated animals were reduced remarkably compared with the controls.There were no significant differences in the mean of the bone mineral density（BMD） of femurs,although in the control group there was less variation in the individual BMD.The body weight of the 125 ng/kg/d group was significantly decreased relative to the controls and there was also a significant reduction in absolute and relative ovary weight.Selenium binding protein 2, Glutathione S-transferase mu type 3,Lrpapl protein,NADPH,and Peptidylprolyl isomerase D were up-regulated,while Prohibitin and N-ethylmaleimide sensitive factor had depressed levels.The severity of the lesions（adipose degeneration,cytoplasmic vacuolization,cell necrosis etc.） increased with the treated concentration.The 125ng·kg^-1·d^-1 group had a significantly lower body weight and higher absolute liver weight relative to the control group.There was also a significant increase in relative liver weight of the TCDD treated groups compared with controls.Complement component 4, Preaploipoprotein A-IV and LMW T-kininogen 1 precursor were up-regulated. Haptoglobin and Complement component 3 were found in TCDD treated groups but not in the control group,while alpha-1-inhibitorⅢprecursor was only present in the control group. ConclusionsThe study give clues to the mechanisms of endocrine toxicity,immunotoxicity, hepatotoxicity and oxidative toxicity mediated by TCDD.Especially,the data provide further insight into the mechanisms by which TCDD disrupts ovarial function by showing which ovarial proteins are affected by TCDD exposure.更多还原