姐妹染色单体差别染色效果相关因素的研究与改进

摘要

Objective To explore the effects of frozen storage time of bromodeoxyuridine (BrdU) solution,storage time of chromosome slides, ultraviolet exposure time and temperature on the differential staining results of sister chromatid, and to figure out whether it is feasible to put the technique as a classroom experiment project. Methods The chromosome slides were prepared by the standard methods of peripheral blood lymphocyte culture and chromosome preparation. The cells were cultured with the BrdU solutions prepared freshly and stored for 10 years at -20℃ .respectively. The chromosome slides prepared freshly and stored at -20℃ for 1,2,3,4,5 weeks were taken for the ultraviolet irradiation at 50,56 and 60℃ for 10,20 and 30 min,respectively,and then stained with Giemsa for 10 min at the room temperature. The effects of the factors on the differential staining were evaluated according to the staining results and colour difference of the chromatids. Results The staining results of chromatids showed no significant difference after incorporating into DNA with BrdU prepared freshly and stored at -20℃ for 10 years,and the most appropriate final concentration of BrdU was 10 μg/ml. Good differential staining results were obtained from the chromosome slides prepared freshly and stored at - 20℃ for 1 - 5 weeks by UV radiation for 10 - 30 min. Conclusion BrdU solution can be stored at - 20℃ for a long-term without affecting the chemical incorporation into DNA. Chromosome slides stored at - 20℃ can be used for sister chromatid differential staining in 5 weeks after preparation. Sister chromatid differential staining is a technique which is tolerant toward the changes of the related factors,and it is entirely feasible as a classroom experiment after the factors are improved.%目的 探讨影响姐妹染色单体差别染色效果的因素,明确作为课堂实验教学项目的可行性. 方法 用常规外周血淋巴细胞培养、染色体制备技术制备染色体标本片,细胞培养过程中分别加入新配制和-20℃保存10年的BrdU溶液.标本制备后分别取新鲜(1d龄)和-20℃冻存1,2,3,4,5周龄的染色体片用紫外线照射,照射条件为50,56,60℃三种温度,10,20,30 min三个时间,然后Giemsa染色10 min.根据姐妹染色单体着色效果与色差评价各因素对差别染色的影响. 结果 在-20℃冻存10年的BrdU与新鲜配制的BrdU掺入DNA后的染色效果无显著差异,终浓度以10 μg/ml为佳.新制备和-20℃保存1-5周的染色体标本片,在50-60℃的片温范围内,紫外照射10-30 min均获得良好的差别染色效果. 结论 BrdU溶液在-20℃长期冻存不影响DNA掺入效果；姐妹染色单体差别染色对相关因素容差性较强,改进后作为课内教学实验项目具有很好的可行性.