硝酸羟胺诱导小鼠淋巴瘤细胞tk基因杂合性缺失分析

Loss of Heterozygosity Analysis in the tk Gene of L5178Y tk^(+/-)-3.2.7C Mouse Lymphoma Cells Induced by Hydroxylammonium Nitrate

目的探讨tk基因分子突变的类型,对不同剂量硝酸羟胺诱导小鼠淋巴瘤L5178Y3.2.7c-tk+/-细胞tk基因突变的杂合缺失进行分析。方法使用硝酸羟胺50～500mg/L剂量对L5178Y细胞进行梯度染毒,分别测定细胞毒性,细胞接种效率,相对悬浮生长率和突变频率。挑选经硝酸羟胺诱导的tk基因突变子(tk-/-)和自发突变体,提取基因组DNA,经等位基因特异性PCR扩增,杂合性缺失(LOH)分析等技术,分析其杂合性缺失的发生。结果大集落和小集落诱导突变体tk基因LOH发生率分别为62.2%和98.9%。由LOH分析的实验结果显示,诱导突变体的LOH在LC与SC之间差异具有统计学意义(P<0.05)。结论功能性等位基因缺失是HAN诱导tk基因分子突变的重要形式之一,在不同集落类型之间可能具有不同的突变机制。

Objective To investigate the effect of hydroxylammonium nitrate (HAN) on the tk gene of mouse lymphoma cells. Methods L5178Y cells were treated with HAN at different concentrations. Determinations of cytotoxicity,cell inoculation efficiency,relative survival rate and mutation frequency were performed. Results Loss of heterozygosity(LOH)analysis employing a microsatellite region within the tk locus revealed that the HAN induced mutants lost the functional tk allele(LOH 62.2%,98.9%). LOH induced by different doses of HAN showed there was significant difference between large colony and small colony. Conclusion HAN has obviously cytotoxic and mutagenic effects on tk gene in L5178Y cells. Functional tk allelic gene loss was a major mutational event in hydroxylammonium nitrate induced tk mutants.