【作者】 王茜；

【导师】 王兰；

【作者基本信息】 山西大学 ， 动物学， 2003， 硕士

【摘要】 长江华溪蟹（Sinopotamon yangtsekiense）广泛分布在我国的淡水水域中，镉、铜对该物种的影响在一定程度上可反映出重金属对水体中十足类甲壳动物的影响。利用最新发展起来的分子毒理学研究技术，采用体外暴露的方式，研究镉、铜对溪蟹的影响。 本实验共分五个部分： 第—部分 镉对长江华溪蟹的急性毒性与积累 采用原子吸收分光光度计，测定镉在溪蟹体内的生物积累。结果表明：镉的生物积累具有选择性，积累的顺序依次为：外壳＞鳃＞肝胰腺＞触角腺＞卵巢；镉对蟹体的作用具有浓度—剂量效应关系；但镉对蟹体的时间—剂量效应关系不很明显。 第二部分 镉对长江华溪蟹精子发生过程中高尔基体超微结构的影响 应用透射电子显微技术（TEM），研究了低浓度镉（1.87mg／L）对溪蟹各级生精细胞超微结构的影响。结果表明：各级生精细胞中高尔基体均有不同程度的变化。随镉处理时间的延长，高尔基体数量增多，扁平膜囊减少，膜囊肿大，膜囊逐渐解体；分泌小泡增多，小泡体积增大。其中，精母细胞表现较为突出，尤其是次级精母细胞。另外，其他的细胞器如细胞核、线粒体、内质网和溶酶体也有不同程度的变化。这些变化使精子发生受阻。 第三部分 镉对长江华溪蟹精子发生过程中细胞核超微结构的影响 应用透射电子显微技术（TEM），研究了低浓度镉（1.87mg／L）对溪蟹各级生精细胞中细胞核超微结构的影响。结果表明：核的形状从正常的圆形或卵圆形，到处理后形状多样；核膜先肿胀膨大，进而破裂，核孔增多。核内染色质也受到影响，分布不均匀或聚集成块，核仁有或无。 第四部分 铜对长江华溪蟹酯酶同工酶的影响 采用聚丙烯酰胺凝胶（PAGE）电泳，活性染色的方法，研究了不同浓度的铜处理后对雄蟹三种组织器官（鳃、肝胰腺、精巢）酯酶同工酶（EST）的影响。结果表明：雄蟹的鳃、肝胰腺、精巢酯酶同工酶都出现了不同程度的变化，鳃酯酶同工酶有2条酶带缺失，相对活性变化不大；肝胰腺和精巢既有新增的酶带又有缺失的酶带，新增酶带相对活性增强，缺失酶带相对活性减弱，说明铜对酯酶同工酶既有激活又有抑制作用，并存在组织差异和剂量效应关系。同时还分析了铜对雄蟹酯酶同工酶影响的可能机理。 第五部分 铜对长江华溪蟹精巢总DNA的损伤 用浓度为2.43mg／L氯化铜（CuCl₂）溶液采用体外暴露的方式处理雄蟹，分别在10d、25d、40d随机取3只雄蟹，先用EDTA和70％乙醇作为固定剂固定，然后采用经典的酚-氯仿法提取精巢总DNA，以总DNA为模板，进行RAPD分析。DNA电泳结果显示：铜处理后的精巢遗传物质未出现断裂。RAPD扩增产物结果表明：铜能使精巢总DNA的条带发生变化。在24条随机引物中，一部分随机引物在对照组及处理组模板DNA的扩增条带出现了差异，并且随着处理时间的延长，RAPD电泳条带的差异也越明显。同时还探讨了材料的固定方法和模板DNA量的确定。更多还原

【Abstract】 The crab Sinopotamon yangtsekiense is widely distributed in Chinese freshwater area. Effects of heavy metal ,such as Cd and Cu,may reflect the effects of the water pollution of heavy metal on the Decapodas (Crustacea:) to some extent Based on the early work, we studied the effects of Cd and Cu on Sinopotamon yangtsekiense by using the new developed technology of molecular toxicology.There are five parts in this experiment:Part one: The Acute Toxicity and Accumulation of Cd2+ in Freshwater Crab S. yangtsekienseMany organisms inclouding the gill, the antennal ,the nepatopancreas, the ovary and the crust were nitrificated,and the contents of Cd2+ was determined by spectrophotometry. The results snowed that Cd2+ was accumulated selectively in S.Yangtsekiense .The bioaccumulation of each organ decreased in turn :the crust>the gill>the hepatopancreas>the antennal gland>the ovary Among them ,the bioaccumulation in gill was 8-50 times higher than that of other organs except for the crust in the same condition. The bioaccumulation of Cd2+ in S.Yangtsekiense had the concentration-effect relationship, that is to say,the more the concentration increased ,the more the bioaccumulation was. But the time-effect relationship was r籺 observed about the bioacoirnulation of the S. Yangtsekiense.Part two: Effects of Cadmium on infrastructure of Golgi Bodies during Spermatogenesis of Freshwater Crab S. yangtsekienseUsing transmission electron microscopy , on effects of low dosage of Cd2+ (1.87mg/L)on ultrastmctrue of Golgi bodies(GB) during spermatogenesis of freshwater crab 5! yangtsekiense at three different times(5d,15d30d), were studied. The results showed that the alterations of Golgi bodies were not obvious in control group and the treated group in the 5th day after an experimental exposure to low dosage of Cd2+, but were very obvious on the 15th and 30th day. Some membranous sac swelling, disintegrated at last During primary and secondary spermatocvte stage, the quantities of GB became more and more. There are more secretive activities on the trans face in the long coure of time , and different alteration in the mitochondria and the rough endoplasmic reticulum(RER). And at the same time the quantities of rysosomes were manifolded During spermatid and spermatozoa stage , owing to GB could not be synemtized with miochondria membrane, and come into being membrane complex. Mitochondrial great swelled and vacuolus. All these changes hindered the spermatogenesis of s. yangtsekiense.Part three: Effects of Cadmium on Ultrastructure of Nucleus during Spermatogenesis in crab S. yongtsekienseStudy effects of low dosage of Cd2+ (1.87mg/L) on ultrastructure of spermatogenic cell nucleus at the different tirnes(5d, 15d, 30d) By transmission electron microscopy(TEM).Showed that the nucleus were affected by the low dosage of Cd2+ during the whole process of Spermatogenesis .First, the nucleus shape varied from spherical or ellipsoidal to all kinds of shapes. Second, nuclear membrane appeared swelling,and disintegrated in the long times, at the same time the quantities of nuclear pores were increased Third, the chromosomes were massed.Part four: Effects of Copper on Esterase Isoenzyme of Freshwater Crab S. yongtsekienseThe effects of different dosages of Cu2+ on the Esterase Isoenzyme(EST)of the gill,the hepatopancreas and the testis were investigated by using polyacrylamide gel electrophoresis (PAGE).The results showed that the effects of Cu2+ on EST isoenzyme activity could be not only activated but inhibited as well There were different effects of Cu2+ on EST isoenzyme of the major tissues. At different dosages of Cu2+, there was the main dosage-effect for the EST isoenzyme of every tissue. From the results it can be concluded that the EST isoenzyme was very sensitive to the toxication of Cu2+ and can be used as a bioindicator of the environmental pollution.Part five: Damage of Copper on the Apermatogenous Genome of crab 51 yongtsekiense Analysed by RAPDEffects of Cu2+ (2.43mg/L) during Sp更多还原