【作者】 曹庆珍；

【导师】 施华宏；

【作者基本信息】 华东师范大学 ， 环境科学， 2011， 硕士

【摘要】 近年来,全球两栖类动物种群衰退现象十分严重,已有研究表明与环境污染物有关。有机锡是目前已知内分泌干扰物质(EDCs)中应用最广的金属化合物,TBT也是人为引入水环境最毒的化学物质之一。两栖类变态实验(AMA)被认为是检测甲状腺激素干扰作用简单而特异的方法,这一方法是在爪蟾蝌蚪变态期间,通过形态学和组织学等指标对污染物是否具有甲状腺激素干扰效应进行检测。而本论文采用分子指标,研究了有机锡化合物对热带爪蟾蝌蚪和胚胎发育过程中相关基因mRNA表达的变化,并探讨了甲状腺激素干扰效应及可能的作用机制。首先,用50和200 ng·L-1的TBT对热带爪蟾NF51阶段蝌蚪进行48和96 h暴露。结果表明,TBT能引起蝌蚪多个甲调基因表达的变化,但基因的表达具有明显的组织差异性。这些基因涉及到甲状腺激素的合成、运输、转化和代谢等多个过程,表明TBT对爪蟾具有甲状腺激素干扰效应。同时,通过对一些相关基因的检测表明TBT可能通过与RXR结合而对蝌蚪产生甲状腺激素干扰效应。其次,TBT对热带爪蟾胚胎进行24、48和72 h三个不同时间段的暴露,结果表明,TBT能引起胚胎多个不同基因表达的变化。这些基因涉及到甲调信号途径,头部发育和神经脊细胞迁移途径等多个过程,表明TBT影响了爪蟾胚胎发育过程。同时,通过对一些甲调基因及其相关基因的检测,从分子水平上说明TBT在爪蟾胚胎期也具有甲状腺激素干扰效应。一些胚胎发育相关基因表达的异常,影响了胚胎发育,导致胚胎畸形。最后,用0.2、1和5μg·L-1TPT对热带爪蟾胚胎进行72 h时间暴露。结果表明,TPT能引起胚胎尾部多个不同基因mRNA表达的变化,初步探讨了TPT引起的胚胎尾部畸形的分子机制,Foxd 3异常表达,可诱导体轴异常,胚胎尾巴弯曲现象可能与其有关。但是,所做基因有限,还需进一步的实验来研究其致畸机制,揭示如鳍为什么变窄等畸形现象。本论文通过多个实验研究了有机锡化合物对热带爪蟾蝌蚪期和胚胎期相关基因表达的变化,从分子水平上证明了有机锡化合物具有甲状腺激素干扰效应,影响了爪蟾发育过程。同时,也研究了有机锡化合物导致眼睛畸形和尾巴弯曲等畸形现象的分子作用机理。由此可见,在现有的AMA实验的基础上增加分子指标,将大大提高TDCs的筛选速度和检测的灵敏性,而且有助于解析污染物的致毒作用模式。更多还原

【Abstract】 In recent years, amphibian populations have declined dramatically. The causes of such declines may be related to environmental pollutants. The organotin compounds are known as endocrine disrupting chemical (EDCs). TBT is considered to be the most toxic substance ever introduced into the marine environment. The Amphibian Metamorphosis Assay (AMA) was conducted to determine the potential thyroid hormone disrupting effect. Morphological and histological endpoints are often used in AMA. In this paper, we used molecular markers to determine the thyroid hormone disrupting effect of two organotin compounds. We studied the effects of organotin compounds on changes of mRNA expressions in development of Xenopus tropicalis tadpoles and embryos, and discussed their endocrine disrupting effects and possible mechanisms.First of all, X. tropicalis tadpoles (NF51) were exposed to TBT for 48 and 96 h. Exposure to 50 and 200 ng-L-1 TBTC1 showed no effects on the survival rate of tadpoles, and suggest that TBT can result in the changes of multiple genes expressions and show high tissue-specific differences. These genes are involved in the synthesis, transportations, transformations and actions, which indicate that TBT shows thyroid hormone disrupting effects. The changes of related genes indicate that TBT might disrupt the thyroid hormone system of tadpoles by binding with retinoid X receptor.Secondly, X. tropicalis embryos were exposed to TBT for 24,48 and 72 h. The results show that TBT can result in the changes of several genes expression. These genes involved in thyroid hormone signal regulating way, head development and migration way of neural crest cell for multiple processes, and show that TBT affect the development process of X. tropicalis embryos. Meanwhile, through detect thyroid hormone receptor genes and their related gene show that TBT also have thyroid hormone disturbance effect in X. tropicalis embryos. Some mRNA expressions abnormalities affected the process of embryonic development, leading to teratogenic effect.Finally, X. tropicalis embryos were exposed to 0.2、1 and 5μg-L-1TPT for 72 h. TPT can induce the changes of many genes expressions in embryonic tail, and primary study its molecular mechanism of malformation. Foxd 3 abnormal expression induces the body axis abnormalities, maybe related to the bending of embryonic tail. But not do so many genes, we still need further experiment to study its teratogenic. mechanism, revealing some abnormal phenomenon such as narrow fm.The changes of related genes indicate that organotin compounds disrupt the thyroid hormone system of tadpoles and embryos and affect the process of development of Xenopus. Organotin compounds might be one of the reasons leading to the decline of amphibian embryos. At the same time, we also studied the molecular mechanism of abnormal eye and bend tail introduced by organictin compounds. Therefore, if we add critical genes’expressions as the markers of AMA, it would not only increase the power to screen the thyroid disrupting chemicals but also provide clues for revealing the mechanisms of contaminants.更多还原