[目的 ]化学物诱发的细胞转化和基因突变分别反映不同的遗传终点。通常的做法是在各自独立的试验中分别进行。本方法介绍了将这两个观察终点结合在同一试验中同步进行的技术 ,以便对其结果进行分析比较 ,为探明化学物遗传毒性的特征 ,以及可能的分子致癌机制提供资料与线索。 [方法 ]使用培养的人成纤维细胞或其他永生化细胞系。使用永生化细胞则便于进一步的成瘤性恶性转化鉴定和突变体的克隆定性。人成纤维细胞维持在补充 10 %小牛血清、抗生素和氢化考的松的Eagle sASP培养液中。细胞自液氮中取出 ,尽快消融复苏 ,增殖为足够的指数生长期细胞群。再按 1× 10 4 细胞 /cm2 的密度接种 ,18h后换以无血清培养液进行化学物处理 ,持续时间 1h。处理后每个浓度至少保证有 1× 10 6个存活细胞。经 8～ 10d突变表达期培养 ,细胞达 80 %汇合时 ,收获合并每剂量组所有细胞 ,同时建立hprt基因突变和转化灶形成试验。其受试细胞总数均为 1× 10 6。诱变试验细胞接种密度为 45 0细胞 /cm2 ,培养液含选择剂 6 硫代鸟嘌呤 ,浓度为 40 μmol/L。培养 2周 ,中间换液 1次。结晶紫染色后计数抗性突变细胞集落数。转化灶试验接种密度为 90 0细胞 /cm2 ,培养液中小牛血清含量为 5 %。培养 6～ 7周 ,每周换液 1次。亚甲蓝? [Objective] Chemical-induced cell transformation and genetic mutations reflect different genetic endpoints, respectively. Common practice is in separate trials separately. This method describes the technique of combining these two observation endpoints simultaneously in the same experiment in order to analyze and compare the results and provide information and clues for the identification of the characteristics of the genotoxicity of the chemical as well as possible molecular mechanisms of carcinogenesis. [Method] Using cultured human fibroblasts or other immortalized cell lines. The use of immortalized cells facilitates further characterization of malignant transformation of the neoplasia and cloning of the mutant. Human fibroblasts were maintained in Eagle sASP broth supplemented with 10% calf serum, antibiotics and cortisol. Remove the cells from the liquid nitrogen, ablation and recovery as soon as possible, proliferation of exponentially growing cell population. Then the density of 1 × 10 4 cells / cm2 inoculation, after 18h replaced by serum-free medium for chemical treatment, the duration of 1h. At least 1 × 10 6 viable cells are guaranteed for each concentration after treatment. After 8 ~ 10d mutation expression period, 80% confluent cells were harvested and all cells in each dose group were harvested, and hprt gene mutation and foci formation assay were established. The total number of cells tested were 1 × 10 6. Mutagenicity test cell seeding density of 45 0 cells / cm2, the culture medium containing a selective agent 6 thioguanine, a concentration of 40 μmol / L. Culture for 2 weeks, the middle fluid 1 time. Count the number of resistant mutant cell colonies after crystal violet staining. The inoculation density of the foci was 90 0 cells / cm2, and the content of calf serum in the culture medium was 5%. Culture 6 to 7 weeks, fluid change 1 times a week. Methylene blue