检测细胞DNA断裂损伤效应的彗星实验法的改良  

Improvements of the Comet Assay for Rapid Detecting the Rupture and Damage of DNA in Cells

作　　者：赖金龙 付倩[1] 任莎[1] 吴国[1] 陶宗娅[1] 张红[1] 罗学刚[2] 

机构地区：[1]四川师范大学生命科学学院,成都610101 [2]西南科技大学教育部生物质材料教育部工程研究中心,绵阳621010

出　　处：《生态毒理学报》2015年第4期195-202,共8页Asian Journal of Ecotoxicology

基　　金：国家核设施退役及放射性废物治理科研重点项目(14ZG6101);四川省教育厅重点课题(14ZA0030);四川省生物质改性材料工程技术研究中心(西南科技大学)开放课题(12zxbk03);四川师范大学校级青年课题(14qn07);四川师范大学实验技术课题(SYJS2014-14)

摘　　要：为了解决彗星实验过程中常出现的脱胶、细胞核分离操作繁琐、重复性低等问题,对彗星实验方法进行了改良,初步建立了彗星实验的快速操作流程。结果显示,通过对载玻片进行预处理,可确保凝胶悬挂均匀;采用改良机械法分离的细胞核浓度适中;以0.5%(w/v)涂层琼脂糖作为基层、以1.5%(w/v)低熔点包埋琼脂糖作为叠加层的"双层凝胶法",辅以"推片法"铺胶,操作便捷且不发生脱胶现象;细胞核膜经裂解处理后再进行电泳和荧光观察,彗星图像清晰,杂质少。应用改良后的彗星实验方法,操作简便,耗时更短,实验效果良好,可快速检测出细胞DNA损伤效应。The comet assay or single cell gel electrophoresis assay （SCGE） provides a simple and effective method for evaluating DNA damage in cells. The method is based on the capability with which the denatured and cleaved DNA fragments migrated out of the nucleoid under the effects of external electric field, whereas undamaged DNA migrated slower and remained mainly in the nucleoid under the same condition. Degree of DNA damage could be assessed by evaluating the tail length of comet （TL）, the percent DNA in the tail （TD） and the tail moment （TM） of the DNA ＂comet＂ which were built upon the DNA fragments. Although the details and the technique process of the method has been improved recently, there are many problems and difficulties which still remain unresolved,such as the agarose gel debonding, very complex results of the nucleus separation, low repeatability of experimental results and non-standardized technique process of the method, and etc. In view of the above problems, the technical details of the quick process method have been improved in the research. The results showed that it was ensured that the agarose gel was distributed uniformly on the slides through the pretreatment of slide; the nucleus concentration were appropriate which were collected by the improved mechanical separation method; the problem of agarose gel debonding were better solved by the way of double gel layer in which 0.5% （weight in volume, w/v） agarose was used as the basal layer and the low metling agarose of 1.5 % （w/v） used as the overlay, and agarose gel laid slowly with coverslip. It is indicated that the clear picture and fewer impurity could be presented by the improved comet assay and the method is a simple and rapid for analysis degree of DNA damage.

关 键 词：铯 蚕豆根尖细胞 遗传毒性 DNA损伤 彗星实验 改良 

分 类 号：Q331[生物学—遗传学]