用BrdU法检测外周血淋巴细胞hprt突变的方法学探讨

Study on 5-Bromodeoxyuridine Labeling Method for Detecting hprt Mutation in Peripheral Blood Lymphocyte

目的 探索用 5 -溴脱氧尿嘧啶 (5 bromodeoxyuridine,BrdU)法检测次黄嘌呤鸟嘌呤磷酸核糖转移酶基因 (hypoxanthine guaninephosphoribosytransferasegene,hprt)突变的操作程序。 方法 用正交设计法就BrdU法的最佳检测条件进行了探讨。结果 本研究所得的最佳操作程序是 :将血样放入 4℃冰箱中冷藏2 4h ;培养基中 6 巯基鸟嘌呤 (6 thioguanine ,6 TG)最终浓度为 1×10 -4mol/L ;BrdU最终浓度用1× 10 -4mol/L ;加入BrdU后培养时间为 16h ;Giemsa染液浓度为 4 % ;染色时间为 15min。结论 BrdU法是一种较好的检测hprt突变的方法。

Objective 5 Bromodeoxyuridine(BrdU)labeling method is important for detecting hprt mutation.The optimum conditions of this method are experimented.Methods Orthogonal design is used in experiments.Results The optimum procedure is as follows:Keep the blood samples in refrigerator at 4℃ for 24 h .Make the final concentrations of both 6 thioguanine(6 TG)and BrdU in culture medium at 1×10 -4 mol/L .Incubate for 16 h.Stain with 4% Giemsa staining solution for 15 min.Conclusions BrdU labeling method is good for detecting hprt mutation.