【作者】 文琛；

【导师】 邬红娟；

【作者基本信息】 华中科技大学 ， 环境科学， 2007， 硕士

【摘要】 孔雀石绿常被用于治陶业、纺织业、皮革业、食品颜色剂和细胞化学染色剂,1933年起作为驱虫剂、杀虫剂、防腐剂在水产中使用。目前孔雀石绿虽已由于其高致癌和高致畸、致突变被许多国家列为水产殖禁用药物,但是孔雀石绿作为一种染料很容易购得,而且流通渠道管理不善导致其禁而不绝。国内外关于鱼体中发现孔雀石绿的消息仍时常见报。目前国内外对孔雀石绿的研究多重在检测水生生物中所含孔雀石绿量,对水体中孔雀石绿浓度对水生生物生活的影响研究较少。本文研究孔雀石绿对鱼细胞的毒性。首先,本文归纳了目前利用鱼细胞进行毒理学研究的概况。其次,讨论了各种检测细胞存活量的方法,确定了MTT检测法的可行性,并且针对不同细胞进行了其最适条件的选择。最后,在上述研究的基础上,运用体外培养细胞技术,进行了孔雀石绿对三种鱼细胞(鲤鱼上皮癌细胞EPC、草鱼肾细胞CIK、斑点叉尾鮰鱼卵巢细胞CCO)毒性的初步研究。对全文进行总结,得出要控制孔雀石绿的污染,必须从控制污染源出发,不但要禁止其作为兽药用于渔业,同时要对排放孔雀石绿浓度做出规定,以保证水质。主要实验结果如下:1)确定三种鱼类细胞MTT法最适条件:其吸收波长皆为570nm;CCO细胞,最佳细胞接种量为5×104个/孔;EPC和CIK这种细胞,最佳细胞接种量为2×104个/孔;MTT的反应时间4小时;MTT终浓度为1mg/ml。2)孔雀石绿对于所研究的三种细胞的毒理研究表明孔雀石绿对于细胞的致死基本一致,超过安全浓度的孔雀石绿会引起细胞变形,由贴壁型细胞特有的梭型变圆,浓度增大还会引起细胞脱落,培养液中出现大片悬浮物的现象,直至孔雀石绿浓度高至致死浓度,使细胞死亡。其中孔雀石绿对CIK细胞的24h安全浓度为0.07mg/L,对CCO细胞和EPC细胞的24h安全浓度分别为0.2mg/L和0.43mg/L。孔雀石绿对这三种细胞的24h半致死浓度分别为:3.73 mg/L、4.12 mg/L、4.43mg/L。比较这三种细胞对孔雀石绿的敏感性,得出结论CIK细胞对于孔雀石绿的作用最敏感。更多还原

【Abstract】 Malachite green has been used extensively for silk, wool, jute, lether and cotton. This dye has been found extensive use all over the world in the fish farming industry as a fungicide, ectoparasiticide and disinfectant since 1933. Due to its potential animal carcinogenicity, mutagenity and teratogenicity, MG has never been registered as a veterinary medicine in the many countries. Because MG is easily available at low cost and highly effective, there is still concern about its illegal use. The study is mostly analysis of MG content in fish. There is little report about the infection of different MG concentration in water.The theme of this paper is the toxicity of malachite green to fish cell. Firstly, the paper summarizes the reports about using fish cell in toxicology. Secondly, the article shows the analysis about methods to study the cell livability. It makes sure the MTT assay is feasible and determine the optimum conditions of this assay. Finally, an attempt has been made to collect information of the toxicity of malachite green by MTT assay. The EPC cell、CIK cell and CCO cell was exposed in normal and malachite green polluted states. The article points out that the control of MG pollution based on controlling the source of this pollution. It is necessarily to forbid MG as a veterinary medicine, at the same time it is necessarily to establish standard for the max concentration of MG in water to ensure water quality.Following are the main results:1) The results showed that the optimal condition of this assay for three fish cell lines: the wavelength was 570nm, the optimum cell number was 5×10⁴/well for CCO cell line, the optimum cell number for EPC cell line and CIK cell line was 2×10⁴/well. The MTT concentration was 1mg/ml and the culture time was 4h.2)The research showed that MG represented same physiological effects to different cell line. Cells would be transmogrification after exposed in MG solution over safe concentration. The livability of cell depended on the concentration of malachite green after 24h. The safe concentration of MG for CIK cell line, CCO cell line and EPC cell line is 0.07mg/L, 0.20mg/L and 0.43mg/L respectively. The IC₅₀ of MG for these three cell lines are 3.73mg/L, 4.12mg/L and 4.43mg/L respectively. Among these cells, CIK cell is the most sensitive to low concentration malachite green.更多还原