[关键词]
[摘要]
目的 建立高通量评价药物遗传毒性的微型细菌回复突变(Ames)试验。方法 采用平皿掺入法进行标准Ames试验,采用6孔板掺入法进行微型Ames试验,两试验均设置阴性对照组和阳性对照组,2组又分别分为代谢活化和非活化2个亚组,各组培养基中分别加入菌液(鼠伤寒沙门菌组氨酸缺陷型菌株:TA97、TA98、TA100、TA102和TA1535),活化组加大鼠肝微粒体酶(S9)混合液,阳性对照组再加入阳性诱变剂:Dexon(-S9),应用于TA97、TA98和TA102;NaN3(-S9),应用于TA100和TA1535;2-AA(+S9),应用于TA97、TA98、TA100、TA102和TA1535。凝固后37℃温箱培养约48 h,计数各组回变菌落数。结果 在标准Ames试验中,阴性对照组各个菌株回变菌落数均在本实验室自发回变菌落数正常值范围内。在两试验中,阳性对照组所诱导的回变菌落数均是阴性对照组的2倍以上,标准试验细菌回变菌落数约为微型试验的5倍。结论 在6孔板上进行微型细菌回复突变试验,大大减少了受试物的用量,提高了筛选速度。本实验室建立的微型细菌回复突变试验背景数据,用于遗传毒理的检测是高效可靠的。
[Key word]
[Abstract]
Objective To establish the method of bacterial reverse-mutation test for high throughput evaluation of drug genotoxicity. Methods The standard Ames test was carried out by the method of plate with incorporation, and the mini-Ames test was performed using 6-well micro plates with incorporation. We established the negative control group and positive control group with metabolic activation or not. The bacteria solution (TA97, TA98, TA100, TA102, and TA1535) was added to the medium respectively. Rat liver microsomal enzyme (S9) mixture was added to the activation group, and the positive control group was added with the positive mutagens:Dexon (-S9) applied to TA97, TA98, and TA102, NaN3 (-S9) applied to TA100 and TA1535, and 2-AA (+S9) applied to TA97, TA98, TA100, TA102, and TA1535. After incubated, culture plates incubated at 37℃ for about 48 h, counting the number of colonies in each group. Results In the standard Ames test, the number of bacterial colonies in the negative control group was all within the normal range of spontaneous colonies in the laboratory. The number of colonies induced by the positive control group was over two times than that of the negative control group. And the number of colonies in classical test was about five times than that of the mini-Ames. Conclusion The mini-Ames test on 6-well micro plates greatly reduced the dosage of the subject matter and improved the screening speed. The background data of mini-Ames test had been established in our laboratory and this test was highly effective and reliable for the detection of genetic toxicology.
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[基金项目]
国家科技重大新药创制项目(2015ZX09501004);天津市科技计划项目(16PTGCCX00090)