[关键词]
[摘要]
目的 探索小鼠淋巴瘤胸腺激酶(tk)位点基因突变试验中血清灭活对结果的影响。方法 取对数生长期的小鼠淋巴瘤细胞(L5178Y3.7.2c-tk+/-)设立阴性对照组和阳性对照组,阴性对照组加入100 μL DMSO,阳性对照组加入100 μL 10 μg/mL 4-硝基喹啉(4-NQO)诱导突变,测定细胞悬浮增长率(SG)、相对悬浮增长率(RSG)、平板效率(PE)、相对存活率(RS)和相对总生长率(RTG)等指标。根据血清灭活与否和选择突变剂三氟胸苷(TFT)的含量,在筛选突变集落过程中,将阴性对照组和阳性对照组各分为:灭活组、未灭活+1.5倍TFT组、未灭活组,测定突变频率(MF),对试验结果进行评价。结果 阴性对照组SG在8~32,细胞倍增速度符合试验要求;PE在65%~120%,符合试验成立条件。阳性对照的灭活组、未灭活+1.5倍TFT组、未灭活组中MF均比阴性对照组至少增加300×10-6且小克隆突变频率至少占40%,符合试验要求。阴性对照灭活组的MF数值在标准范围内,未灭活组的MF数值明显高于灭活组,超过标准数值范围,且96孔板中的大小克隆不易区分;阴性对照未灭活+1.5倍TFT组使得MF数值在标准范围内,但远高于灭活组。结论 在小鼠淋巴瘤试验中,测定MF的平板一定要用灭活的血清,未灭活血清可能通过抵消TFT作用对结果产生很大影响。
[Key word]
[Abstract]
Objective To explore the effect of inactivated serum on the mouse lymphoma tk gene mutation assay. Methods The negative control group and positive control group were set up for the mouse lymphoma cells (L5178Y3.7.2c-tk+/-) during the logarithmic growth period. The negative control group was added to 100 L DMSO. The positive control group added 100 μL 10 μg/mL 4-nitroquinoline (4-NQO) induced mutation, and the cell suspension growth rate (SG), the relative suspension growth rate (RSG), plate efficiency (PE), relative survival rate (RS), relative total growth rate (RTG), and other indicators were measured. According to the inactivation of serum and the content of three fluorothoracoside (TFT), the negative control group and the positive control group were divided into inactivation group, not inactivated + 1.5×TFT group and the not inactivated group. We measured the mutation frequency (MF) value and evaluated the test results. Results In negative control group, SG was from 8 to 32, and cell doubling speed accords with test requirement. PE from 65% to 120% conforms to the established condition of experiment. MF in positive control group increased by 300×10-6 than the negative control group, and the frequency of the small clone mutation was at least 40%, which was in line with the test requirements. MF value of the negative control inactivation group was within the standard range. The MF value of the not inactivated group was significantly higher than that of the inactivated group, exceeding the standard number, and Banzhong size clones were not easy to distinguish; The negative control not inactivated +1.5×TFT group made the MF value within the standard range, but much higher than the inactivated group. Conclusions In mouse lymphoma test, the plates with measuring mutation frequency must use the heat inactivate serum, and the not inactivated serum may have a great effect on the results by counteracting TFT.
[中图分类号]
[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)