【摘要】 用MNNG处理HL-60细胞和8-AG软琼脂集落形成方法,成功地筛选出一株抗高浓度8-AG和完全缺失HGPRT活性的人早幼粒白血病细胞突变株,命名为HL-60-AR。该株细胞能抵抗8-AG的毒性作用,对HAT选择培养基十分敏感,检测不到HGPRT活性,不能参入~3H-次黄嘌呤。HL-60-AR细胞基本上保持了野生型HL-60细胞的生物学特征。突变细胞已在体外培养11个月,传100多代,遗传标记仍然稳定。更多还原

【Abstract】 A human promyelocytic leukemia cell mutant (HL-60-AR) with deficiency in hypoxanthine guanine phosphoribosyltransferase (HGPRT) and resistance to 8-AG was established by treatment of the HL-60 cells with 2μg/ml N-methyl-N/-nitro-N-nitrosoguanidine(MNNG), and prescreened in 0.3% agar RPMI-1640 semisolid medium containing 20μg/ml 8-AG.The mutant cells show the following characteristics: 1) they are resistant to cytotoxic action of 8-azaguanine (8-AG)and can grow vigorously in medium containing 20μg/ml 8-AG;2) the mutant cells can form colonies in soft agar medium in the presence of 8-AG;3) they are highly sensitive to HAT selective medium and fail to grow in HAT medium or form colonies in soft agar medium containing HAT; 4) quantitative assay shows that the mutant cells are completely deficient in HGPRT activity;and 5) radioautography has showed clumps of radioactive silver grains over the HL-60 cells, while in the mutant cells no silver grain can be discernible due to the absence of 3H-hypoxanthine incorporation. Besides, the mutant cells maintain their parental HL-60 cells biological characteristics .Up to now, they have been cultured for more than 11 months and have been passed for more than. 100 passages in vitro., This mutant cell line will certainly provide an invaluable model to explore cellular engineer-ing on regulation of human tumor cell malignancy.更多还原