双酚A与四溴联苯醚（BDE-47）暴露对紫贻贝组织病理与生殖相关基因表达的研究

	双酚A与四溴联苯醚（BDE-47）暴露对紫贻贝组织病理与生殖相关基因表达的研究
	章盈盈
学位类型	硕士
导师	吴惠丰 ; 赵建民 ; 曲江波
	2014-05-21
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	环境工程
关键词	内分泌干扰物紫贻贝 17β-羟基类固醇脱氢酶卵黄蛋白原基因克隆基因表达组织病理
摘要	近年来，海洋污染日益严重，海洋环境中的内分泌干扰物对海洋生物及海洋生态系统的影响，已成为近年来的研究热点。软体动物作为生物界第二庞大的生物类群，在生态系统中扮演着重要的角色，同时也常被用作生态健康评价的指示生物。目前，对于软体动物生殖和内分泌系统的研究甚少，虽然一些研究揭示了脊椎动物的性激素同样存在于软体动物中，但是关于这些性激素的合成通路及参与这些通路的酶类还亟待研究。17β-羟基类固醇脱氢酶是一类多功能的酶，参与了性腺甾醇、脂肪酸、类视黄醇和胆汁酸等物质的代谢。卵黄蛋白原是卵黄蛋白的前体，通常只在性成熟的雌性中表达，但是在外源激素作用下，在雄性及幼体中也可能表达。本研究选取紫贻贝作为研究对象，采用RACE等方法克隆到两种类型17β-羟基类固醇脱氢酶基因的全长序列与卵黄蛋白原的部分序列，并选择了典型的内分泌干扰物BPA和BDE-47作为暴露物，结合实时荧光定量PCR技术与组织病理学等技术，探讨了内分泌干扰物对紫贻贝内分泌系统的影响，从而为表征内分泌干扰效应敏感指标的筛选提供科学依据。本论文获得如下结果： ⑴基因的克隆与序列分析 10型和12型17β-羟基类固醇脱氢酶的基因全长为916 bp和1196 bp，分别编码259和325个氨基酸。两种基因推断的氨基酸序列都含有保守的TGXXXGXG 结构域，催化三联体结构域（丝氨酸 - 酪氨酸 - 赖氨酸），保守的YXXXK活性结构域，其中酪氨酸作为活性催化碱基。克隆获得的卵黄蛋白原片段长为712 bp，位于保守的卵黄蛋白原结构域内。系统进化树分析结果表明，10型和12型17β-HSD所处的位置与其分类学位置相符合，10型17β-HSD与8型17β-HSD进化上关系较近，12型17β-HSD与3型17β-HSD关系较近。 ⑵基因的组织分布特征组织分布结果显示，两种类型的17β-羟基类固醇脱氢酶均呈多组织分布特征，主要表达于性腺与消化腺组织，而卵黄蛋白原基因则主要表达于雌性贻贝的血细胞、消化腺和性腺，在雄性中几乎不表达。 ⑶典型内分泌干扰物的暴露下基因表达变化急性暴露实验结果显示，BPA及BDE-47暴露均显著降低了两种类型17β-HSDs基因的表达。在雌雄贻贝中，两种基因的表达量在24 h显著减少（P<0.05），随着暴露时间的延长，在96 h雌性贻贝逐步恢复到对照水平，而雄性贻贝还处于显著抑制的状态。雌雄贻贝卵黄蛋白原基因在BPA及BDE-47暴露下，表达量均出现先上升后下降的趋势，各处理组的基因表达量在48 h均发生显著变化（P<0.05）。亚慢性暴露实验的结果与急性暴露结果类似，受到上述污染物暴露后，17β-羟基类固醇脱氢酶基因的表达量出现下降，而卵黄蛋白原基因表达量出现上升。同时，亚慢性暴露结果表明暴露剂量与检测效应之间存在一定的关系，即高浓度处理组对上述基因的诱导效果比低浓度处理组更显著。 ⑷典型内分泌干扰物的暴露下组织病理分析组织学分析结果表明，BPA和BDE-47均能对紫贻贝的性腺、鳃和消化腺组织造成损伤，且随着浓度的升高，损伤的程度加大。造成的损伤包括使卵巢滤泡体积变小，数量减少，影响卵母细胞的发育，影响雄性贻贝的精细胞发育；鳃组织的纤毛断裂扭曲，上皮细胞肿胀，鳃叶萎缩变短，鳃丝断裂；消化腺组织的细胞肿胀严重，胞质溶解造成局部坏死，大范围内出现空泡化现象。
其他摘要	Currently, the pollution of the marine environment is becoming increasingly serious. The water environment has become a major concern of society due to its role as the biggest depositary of environmental pollutants, especially environmental endocrine disrupting chemicals. Mollusks are the largest animal phylum, they are ecologically important and considered excellent indicators of ecosystem health. So far, few studies have demonstrated the reproductive and endocrine system of mollusks. Although some studies reported the presence of (vertebrate-type) steroids in mollusks, information regarding enzymatic pathways involved in steroid synthesis and further catabolism of those steroids is still fragmentary. 17β-Hydroxysteroid dehydrogenases (17β-HSDs) are multifunctional enzymes involved in the metabolism of steroids, fatty acids, retinoids and bile acid. Vitellogenins (Vtg) are the major precursors of the egg-yolk proteins, vitellins (Vn), which were only expressed in sexually mature females in oviparous organisms. However, vitellogenins may also be expressed in males and juveniles in response to exogenous estrogens. In this study, we cloned two novel 17β-HSDs using rapid amplification of cDNA ends (RACE) approaches and partial sequence of vitellogenin through RT-PCR from Mytilus galloprovincialis. And the expression level of the 17β-HSDs and vitellogenin transcripts in digestive glands, as well as routine tissue histology the were studied after exposed to endocrine disruptors (Bisphenol A or 2,2′,4,4′-tetrabromodiphenyl ether). The studies we conducted here were to investigate the effect caused by the typical endocrine disrupting chemicals and therefore try to screen the sensitive biomarkers. The results of the studies are as follows. ⑴Gene cloning and sequence analysis The full-length of MgHsd17b10 and MgHsd17b12 were 916 bp and 1196 bp, encoded a polypeptide of 259 and 325 amino acids, respectively. The deduced animo acid sequence revealed the presence of a conserved N-terminal co-factor binding site motif (TGXXXGXG), a putative catalytic site (YXXXK) and catalytic triad of Ser-Tyr-Lys. The partial sequence of MgVtg contained 712 bp, which located within the conserved vitellogenin domain. Phylogenetic analysis revealed that MgHsd17b10 and MgHsd17b12 were evolutionarily clustered with other invertebrate 17β-HSD type 10 and 17β-HSD type 12 homologues. 17β-HSD type 10 and type 12 seemed to have a close evolutionary relationship with 17β-HSD type 8 and 17β-HSD type 3, respectively. ⑵Tissue distribution of the genes The MgHsd17b10 and MgHsd17b12 transcripts could be detected in all examined tissues with higher expression levels in digestive glands and gonad, while vitellogenin was mainly expressed in hemocytes, digestive glands and gonad of female mussels. ⑶ Effects of typical EDCs exposure on gene transcription The expressions of MgHsd17b10 and MgHsd17b12 transcripts were both down-regulated significantly in digestive glands after exposed to BPA/BDE-47 for 24 h (P<0.05). As exposure time increased, the expression levels of these genes in the females restored to that of the control groups, while the levels of these genes in the males were still inhibited. The mRNA expression level of vitellogenin significantly increased at 48 h exposure (P<0.05). Similar results were also found in sub-chronic (28 d) exposure. Moreover, the dose-effect relationship seemed to be found in the sub-chronic exposure. ⑷ Histological analysis after the exposure to EDCs Our histological examination showed the damaging effects of BPA and BDE-47 on tissue structure and status. And the effect was in correlation to the concentration of the chemicals. The ovary showed atretic ovocytes, and the follicles were reduced in size and number. Furthermore, these chemicals caused disruption of gametogenesis in male mussels. As for the gill tissue, the cilium was damaged and epithelial cells puffed up. Overlap and atrophy of nuclei as well as shortening of gill-filament was found. Hepatocytes tumefied and nucleolus changed in size. Vacuolation occured in wide-range.
语种	中文
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/6811
专题	中国科学院烟台海岸带研究所知识产出_学位论文
推荐引用方式 GB/T 7714	章盈盈. 双酚A与四溴联苯醚（BDE-47）暴露对紫贻贝组织病理与生殖相关基因表达的研究[D]. 北京. 中国科学院研究生院,2014.

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