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目的 探讨乙基亚硝基脲 (ENU)诱导人次黄嘌呤鸟嘌呤磷酸核糖基转移酶 (HPRT)基因突变的分子图谱和发生机制。方法 采用单细胞克隆培养、双向筛选计数、多重聚合酶链反应扩增和电泳分析等方法。结果 随着ENU剂量的增加 ,细胞接种存活率非常显著下降 (10 0～ 2 0 0 μg/ml剂量组 )、突变频率显著升高 (12 5～ 2 0 0 0 μg/ml剂量组 )。自发突变没有全部基因外显子缺失型 ,只有 7 7%检测出单个外显子缺失 ;而ENU诱发突变中却有 79 7%显示缺失改变 ,其中缺失突变可以发生于HPRT基因的每个外显子 ,且诱发突变中大多数是多个外显子连锁缺失 (88 1% )。结论 ENU诱发HPRT基因突变图谱与自发突变完全不同 ,易诱发较大遗传结构改变 ,提示其具有较强的致突变作用
Objective To investigate the molecular patterns and mechanisms of human hypoxanthine guanine phosphoribosyltransferase (HPRT) gene mutation induced by ethyl nitrosourea (ENU). Methods Single cell clone culture, two-way screening and counting, multiple polymerase chain reaction amplification and electrophoresis analysis methods. Results With the increase of ENU dosage, the survival rate of cell seeding was significantly decreased (100 ~ 200 μg / ml dose group), the mutation frequency was significantly increased (125 ~ 20 000 μg / ml dose group). In spontaneous mutation, there was no deletion of all exons in the gene, only 7 7% of them detected a single deletion of exon. However, 79 7% of ENU induced mutations showed deletion deletion, of which deletion mutation could occur in every exon of HPRT gene The majority of mutations in the mutagenesis were multiple exon deletions (88 1%). Conclusion The pattern of HPRT gene mutation in ENU is completely different from that of spontaneous mutation, and it is easy to induce large genetic structure change, suggesting that it has a strong mutagenic effect