【作者】 凌发忠；

【导师】 徐忠东；

【作者基本信息】 安徽大学 ， 生物化学与分子生物学， 2013， 硕士

【摘要】 单细胞凝胶电泳(single cell gel electrophoresis, SCGE)是近年来经过不断完善逐步发展起来的一种快速检测单细胞水平DNA损伤的新技术。若细胞DNA受损时,其断片会进入凝胶中,电泳时受损片段向阳极迁移,形成荧光拖尾现象,形似彗星,因此又称彗星实验(comet assay)。细胞DNA受损愈严重,产生的DNA片段就愈多,在电场作用下迁移的DNA量也就越多,表现为尾长的增加和尾部荧光强度的增强。通过测定DNA迁移部分的光密度或迁移长度可定量地统计分析各组细胞的DNA损伤程度。由于彗星实验简便快速、需样品量少、可检测多种毒性物质和适用于任何细胞类型等优点,广泛应用于水质监测。嗜热四膜虫Tetrahymena thermophila)作为生物材料,具有培养简单,生活周期短,对环境的污染物敏感等特点。本实验运用嗜热四膜虫,结合实验的改良使得实验过程变得更加简便,从而建立快速准确评价工业废水的污染状况的一种检测方法。研究内容主要是通过实验证明自制模板铺胶可以取代传统铺胶。改良的嗜热四膜虫彗星实验,与流式细胞术联合检测,以尾长、尾距、olive尾距等指标综合分析医药工业废水(氯代废水、吸附废水)对四膜虫DNA损伤情况,并且比较二者遗传毒性以及嗜热四膜虫彗星实验在其他种类工业废水毒性检测中的应用。本文得出的实验结论如下：(1)标准彗星实验(传统铺胶)与改良彗星实验(自制模板铺胶)按常规方法操作,即培养瓶内细胞达对数期时,分别加入不同浓度的受试物(过氧化氢),同时设相同体积的双蒸馏水作为阴性对照组。暴露一段时间后,离心收集制成一定浓度的细胞悬液,然后制片、裂解、解旋、电泳、染色,在荧光显微镜下观测结果。在相同暴露时间内,同一H2O2浓度下DNA损伤从尾长、尾距、Olive尾距上保持一致。由此可见,自制电泳槽可以取代磨砂载玻片的铺胶法。(2)改良彗星实验对医药工业废水的检测,结合流式细胞术,提高检测结果的准确性。结果表明,该实验改良可以提高效率和稳定性,不同稀释倍数的医药工业废水所引起四膜虫细胞DNA损伤之间呈现良好的剂量-效应关系,这个结果与流式细胞术的结论一致。在稀释相同倍数的情况下,吸附废水的遗传毒性大于氯代废水。(3)本文利用改良彗星实验,对其他四类工业废水(苯甲酸废水、苯甲腈废水、苯甲酸苄酯、氯碱废水)进行了相应的检测。结果表明,四种工业废水引起四膜虫DNA损伤的程度是不同的,其中四种废水对嗜热四膜虫DNA毒性的强弱为：苯甲腈废水>苯甲酸废水>苯甲酸苄酯废水>氯碱废水。以四膜虫为生物材料结合本实验的改良,使得实验过程中样品的预处理和流程变得更加简单。因此,嗜热四膜虫慧星试验改良对于完善环境检测系统和监测环境突变／致癌物的污染具有重要意义。更多还原

【Abstract】 With the development of technology for rapid detection of single cell DNA damage, single cell gel electrophoresis gradually became a new technology in recent years. If the cell DNA damaged, the fragment could get into the agarose gel. The damaged fragment migrated towards the anode after gel electrophoresis, and formed fluorescence tailing phenomenon which was like a comet. So it was also called comet assay. If cell DNA was more damaged, the more fragments would appear. The amount of DNA migration under the electric field was also more, and increased the length and fluorescence intensity of the tail. By measuring fluorescence intensity of DNA migration section or migration length, the technique could analysis the degree of injury of DNA cells of each group. The comet assay was simple and convenient. The experiment only needed small sample volume, which can detect a variety of toxic substances and was applicable to any cell type. At present, it was widely used in water toxicity monitoring.As a model organism, Tetrahymena thermophila had many advantages, such as simple cultivation, short life cycle, and high environmental contaminant sensitivity. This experiment used Tetrahymena thermophila, and combined with the experimental improvement, which made the experiment easier. The experimental improvement established a method of detecting industrial wastewater toxicity, which become rapid and accurate. It had the following content. Experiments proved that the improved method could replace the traditional method. Tetrahymena thermophila comet assay, combining with flow cytometry, analyzed DNA damage of Tetrahymena thermophila by the pharmaceutical industry wastewater, and compared adsorption wastewater with chlorinated wastewater about genetic toxicity. Meanwhile, the technique detected water quality of other industrial wastewater.The conclusions of this experiment were the following.(1) Standard comet assay and modified comet assay operated according to the conventional method. When the cells reached logarithmic phase, added different concentrations of test substances (hydrogen peroxide), at the same time set double distilled water with the same volume as the negative control group. After a period of time, centrifugal collectors made a certain concentration of cell suspension. Then did comet assay and observed results. At the same time of exposure, two methods showed that DNA damage about tail length, tail moment and olive tail moment, was basically consistent. Thus, the self-made template can replace the traditional method.(2) Tetrahymena thermophila comet assay, combining with flow cytometry, analyzed DNA damage induced by the pharmaceutical industry wastewater, which can improve the accuracy of test results. The results showed that the experimental improvement can improve the efficiency and stability, and showed a good dose-effect relationship between dilution ratio and Tetrahymena thermophila DNA damage. This result is consistent with the flow cytometry. In the same case of dilution multiple, genetic toxicity of adsorption wastewater was more than chlorinated wastewater.(3) Improvement of experiment detected other industrial wastewater, such as benzoic acid wastewater, benzonitrile wastewater, benzyl benzoate wastewater and chlor-alkali wastewater. Research showed the four kinds of industrial wastewater had different degrees of DNA damage on Tetrahymena thermophila. The toxicity intensity was:benzonitrile wastewater>benzoic acid wastewater> benzyl benzoate wastewater> chlor-alkali wastewater.In this study, improvement of experiment made the pretreatment and the process simpler. Therefore, Tetrahymena thermophila comet assay had the vital significance for improving the environmental monitoring system and monitoring of environmental catastrophe/carcinogen pollution.更多还原