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目的 为研究丙烯酰胺(AA)致基因突变的机制提供实验依据。方法 挑选经AA诱导的小鼠淋巴瘤L5178Y3.2.7c tk+/-细胞tk基因突变子(tk-/-),提取基因组DNA,用等位基因特异性聚合酶链反应(PCR)扩增、杂合性缺失(LOH)分析、序列分析等技术,分析其突变谱。结果 AA诱导突变体tk基因LOH发生率为78.8%。LOH的发生率随AA剂量的增加呈上升趋势,染毒剂量越大,tk等位基因缺失的发生就越严重。在大集落(LC)中,AA低剂量组(150、300μg/ml)诱导LOH的发生率(25. 0%、33.3%)与自发突变组和AA高剂量组(600、750μg/ml) LOH的发生率(66. 7%、77. 8%、85.7%)相比,差异均有统计学意义(P<0.05)。序列分析证实,AA诱导的tk基因点突变主要为碱基置换以及1个移码,其中碱基置换主要的类型为T:G→G:T的颠换。结论 AA诱导的tk基因突变谱是以功能性等位基因缺失为主的突变类型,其次是碱基置换,移码较少发生。
Objective To provide experimental evidence for studying the mechanism of acrylamide (AA) gene mutation. Methods Mutants (tk - / -) of tk gene in mouse lymphoma L5178Y3.2.7c tk +/- cells induced by AA were selected and genomic DNA was extracted and amplified by allele specific polymerase chain reaction (PCR) Loss of identity (LOH) analysis, sequence analysis and other techniques, analysis of its mutation spectrum. Results The incidence of LOH in tk gene induced by AA was 78.8%. The incidence of LOH increased with the increase of AA dose. The larger the dose, the more serious the deletion of tk allele. In large colonies (LC), the incidence of LOH (25.0%, 33.3%) in AA low dose group (150,300μg / ml) was significantly higher than that in spontaneous mutation group and AA high dose group (600,750μg / ml) (66.7%, 77.8%, 85.7%), the differences were statistically significant (P <0.05). Sequence analysis confirmed that point mutation of tk gene induced by AA was mainly base substitution and one frame shift. The main type of base substitution was T: G → G: T transversion. Conclusion The mutation of tk gene induced by AA is a type of mutation mainly based on functional allele deletion, followed by base substitution, with less frame shift.