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[摘要]
目的 使用L5178Y细胞分别开展tk基因突变试验和hprt基因突变试验评价纳米氧化铁颗粒(iron oxidenanoparticle, IONP)的潜在基因突变风险,比较两种方法的灵敏性。方法 不同质量浓度的PEG-IONP(31.25、62.5、125、250、500 μg/mL)分别与细胞作用3 h,于给药后第0、2、6天将细胞接种于96孔板继续培养9~10 d,用于计算平板接种效率。分别在给药后第2天或第6天加入三氟胸苷(TFT)和6-硫鸟嘌呤(6-TG)作为tk基因和hprt基因选择剂,继续培养14 d后分析基因突变频率。试验平行设置灭菌注射用水溶媒对照组和甲基甲烷磺酸酯(MMS,10 μg/mL)阳性对照组。结果 溶媒对照组和阳性对照组的hprt基因突变率均低于tk基因突变率,且统计学结果提示hprt基因突变试验数据获得显著性差异的起始浓度(250 μg/mL)高于tk基因突变试验(125 μg/mL)。但整体而言两种试验方法对PEG-IONP的评价结果一致。结论 PEG-IONP可引起小鼠淋巴瘤L5178Y细胞tk基因及hprt基因突变率显著性升高。该研究结果可为纳米材料基因突变风险评价的选择提供借鉴与参考。
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[Abstract]
Objective To perform tk gene mutation assay and hprt gene mutation assay using L5178Y cells for evaluating the gene mutation risk of IONP, and comparing the sensitivity of both assays. Methods Cells were treated with different concentrations of PEG-IONP (31.25, 62.5, 125, 250, 500 μg/mL) for 3 h. Cells were seeded in 96-well plates on 0, 2 and 6 days after treatment and cultured for 9 to 10 days for calculating the plating efficiency. TFT and 6-TG were adopt as the tk gene and hprt gene selection agents on 2 or 6 days after treatment respectively, and the gene mutation frequencies were analyzed after a 14 days incubation. Sterile water and MMS (10 μg/mL) were included in parallel as vehicle control and positive control groups. Results The mutation rates of hprt gene in vehicle control group and the positive control group were lower than that of the tk gene, and the statsistical results indicated that the initial concentration for positive result in the hprt gene mutation assay (250 μg/mL) was higher than the tk gene mutation (125 μg/mL). However, the results for PEG-IONP are generally consistent in both assays. Conclusion PEG-IONP could significantly increase the mutation rates of both tk gene and hprt gene in mouse lymphoma L5178Y cells. This data provided reference for the methods selection on the gene mutation risk assessment of nanomaterials.
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[基金项目]
国家十三五“重大新药创制”专项课题(2018ZX09201017);国家自然科学基金(81503347)#共同