摘要
彗星试验方法的初步研究
彗星试验(Comet assay)又称单细胞凝胶电泳试验(SCGE)，是一种快速、灵敏的检测单个细胞DNA断裂的新技术，由于其具备其它试验无可比拟的优越性，因此应用前景十分广泛。为了在本实验室建立该方法，扩展该方法在农药安全性评价中的应用，本研究采用公认的DNA断裂剂重铬酸钾溶液为试验材料，设5个试验剂量，分别为12.5、25.0、50.0、100.0、500.0μmol/L, 处理CHL细胞2个小时，然后对CHL细胞进行单细胞凝胶电泳试验。从细胞的彗星图和试验数据分析可以看出，随着重铬酸钾溶液浓度的增大，彗星尾长明显增长，说明重铬酸钾溶液剂量越大，对CHL细胞DNA的损伤越严重，且不同浓度重铬酸钾溶液与CHL细胞DNA损伤之间存在着明显的剂量－效应关系。这些结果表明在本实验室条件下基本建立起来了该方法，并且找出了适合本实验室条件下的CHL细胞彗星试验方法的条件：
最合适的制片条件，改良的细胞染毒方式，受试物处理CHL细胞时间至少为2小时，细胞裂解时间为2~2.5小时，DNA解旋时间为30分钟，在电压18~25V、电流200~300mA下，电泳20~30分钟。
The Comet assay, also known as the single cell gel electrophoresis (SCGE) assay, is a rapid, visual, and highly sensitive technique for measuring DNA breakage in individual cells. The technique is rapidly becoming a widely used analytical procedure because of its unique advantages. The objective of this study was to adapt the Comet Assay tech-nique to the test conditions of our laboratory. In this study, CHL cells were treated for 2 h with K2Cr2O7 solution at five different concentrations (12.5, 25, 50, 100, and 500 μmol/L). DNA damage was then determined using the Comet assay. Comet graphs of the cells together with associated data indicated that comet tail length increased as K2Cr2O7 concentration increased, thereby showing a dose-response relationship. Thus, the Comet assay was successfully adapted to the conditions of our laboratory. Optimum treatment conditions included the following: optimum conditions of slide preparation ; CHL cell treatment period at least 2 hr; CHL cell lysis period of 2 to 2.5 h; alkali unwinding period of 30 min; electrophoresis conducted for 20 to 30 min at 25 V and 300 mA.
引文
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