乙基亚硝基脲诱导小鼠淋巴瘤细胞tk基因杂合性缺失

目的探讨tk基因分子突变的类型,对不同剂量乙基亚硝基脲（ENU）诱导小鼠淋巴瘤L5178Y3.2.7c-tk^＋/-细胞tk基因突变的杂合性缺失（LOH）进行分析,为环境致突变剂检测和突变机制研究提供依据。方法使用乙基亚硝基脲10μg/mL和100μg/mL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、相对存活率、相对悬浮生长率和突变频率等。挑选经ENU诱导的tk基因突变子（tk^-/-）和自发突变体,提取基因组DNA,应用等位基因特异性PCR扩增、杂合性缺失分析等技术,分析其杂合性缺失的发生。结果低剂量和高剂量诱导突变体的tk基因LOH发生率分别为73.5%和69.1%。LOH分析结果显示,诱导突变体的LOH在大集落与小集落之间差异具有显著性（P〈0.05）。结论功能性等位基因缺失是ENU诱导tk基因分子突变的重要形式之一,不同剂量可能具有不同的突变机制。

LOSS OF HETEROZYGOSITY OF tk GENE IN MOUSE LYMPHOMA CELLS INDUCED BY ETHYLNITROSOUREA

Objective To investigate the mutagenic effect and the loss of heterozygosity（LOH） of tk gene of mouse lymphoma cells induced by ethylnitrosourea（ENU）, so as to provide the basis for detection of environmental mutagenesis and further study of its mechanism. Methods L5178Y 3.2.7c - tk^＋/- cells were treated with different concentra- tions of ENU （ 10 μg/mL, 100μg/mL）. Determination of cytotoxicity, cell plating efficiency, relative survival rate, relative suspension growth rate, and mutation frequency etc. were performed. DNA was extracted from selected mutant gene. LOH was analyzed after PCR of mutant allele. Results The rate of LOH of tk mutant gene induced by low, and high concentration of ENU was 73.6%, and 69.1% respectively. There was significant difference（ P 〈 0.05） between large colony and small colony. Conclusion ENU has obvious cytotoxic and mutagenic effects on tk gene in L5178 cells. Functional tk allelic gene loss may be an important mutational event in ENU-induced mutation and the mechanism may be different between different concentrations.