标准化Ames波动试验最佳条件的优选  

Optimization of standardized Ames fluctuation test

作　　者：陈颖[1] 李莹[1] 管隽[1] 王忠辉[1] 贾玉玲[1] 周莉[1] 孙祖越[1] 

机构地区：[1]上海市计划生育科学研究所药理毒理学研究室,中国生育调节药物毒理学检测中心,上海200032

出　　处：《癌变．畸变．突变》2014年第3期199-203,共5页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：十二五"重大新药创制"科技重大专项(2011ZX09301-005);上海市实验动物创新行动计划项目(11140901300;11140901303);上海市人才发展基金(2010031)

摘　　要：目的:建立Ames波动试验的标准化方法,为其应用于化合物的遗传毒性检测奠定基础。方法:应用4种已知具有遗传毒性的阳性化合物敌克松(Dexon)、叠氮钠(SA)、环磷酰胺(CP)和2-氨基芴(2-AF)进行Ames波动试验。比较2种不同的增菌方法和2种指示剂,建立Ames波动试验的方法。Dexon采用0.1、1和10μg/mL,SA采用0.01、0.1、1μg/mL在非活化条件下与相应的溶剂对照进行试验;CP采用10、100、1 000μg/mL,2-AF采用0.1、1和10μg/mL在活化条件下与相应的溶剂对照进行试验;观察记录阳性孔数,根据情况调整阳性化合物的剂量,直到经统计分析后阳性孔数与溶剂对照组相比,差异有统计学意义(P<0.01)。确定各阳性化合物的剂量后,在相同剂量下重复试验3次,保证试验系统稳定可靠。结果:确定了Ames波动试验4种阳性化合物的剂量。在不加S9代谢活化系统的条件下,Dexon 3μg/mL和SA 0.05μg/mL,3块平行96孔板与溶剂对照组相比差异均有统计学意义(P<0.01),对TA100呈现出强致突变作用;在加入S9代谢活化系统的条件下,CP 100μg/mL和2-AF 30μg/mL,3块平行96孔板与溶剂对照组相比差异均有统计学意义(P<0.01),对TA100也呈现出强致突变作用。同时,在实验过程中改进了试验方法:包括对受试物剂量的标准化,改进了增菌培养的方法,并确定了指示剂。结论:建立了Ames波动试验方法,对已知阳性化合物测定结果的稳定性良好,并在研究中优化了实验条件。OBJECTIVE： We developed a standard Ames fluctuation test in our laboratory and lay the foundation for its application in detecting the genetic toxicity of compounds. METHODS：We used four positive compounds in the test,Dexon,SA,CP and 2-AF. We compared two ways of enrichment culture and two indicators to eatablish the sandand percedure. We adopted 0.1,1,10 μg/mL Dexon and 0.01,0.1,1 μg/mL SA in the non-activated condition with their menstruum as negative control;10,100,1 000μg/mL CP and 0.1,1,10μg/mL 2-AF in the activated condition with their menstruum as negative control. We recorded the number of positive wells,adjusting the dose of the positive compounds accordingly,until there was a significant difference （P〈0.01） between the positive group and the solvent compared group. After determining the dose of the positive control compounds,we repeated the test at the same dose 3 times,to ensure that the test system was stable and reliable. RESULTS：We determined the doses of the four positive compounds for the Ames fluctuation test in our laboratory,Dexon 3 μg/mL,SA 0.05μg/mL,CP 100μg/mL and 2-AF 30μg/mL. In the non-activated condition 3μg/mL Dexon and 0.05μg/mL SA of the 3 96-well plates were significantly different from the solvent control group,exhibiting strong mutagenic effect to the TA100（P〈0.01）. Similarly,100μg/mL CP and 30μg/mL 2-AF in the activated condition of the 3 96-well plates were significantly different from the solvent control group,also showing strong mutagenic effect to the TA100（P〈0.01）. We also improved the test method during the experiment,including sandardizing the doses of test substance,improving enrichment culture method and determining the indicator. CONCLUSION：We established the Ames fluctuation test in our laboratory,confirmed that the known positive compounds were good and stable,and we optimized the experimental conditions in the study.

关 键 词：Ames波动试验 诱变试验 标准化 遗传毒性 

分 类 号：R965.3[医药卫生—药理学]