六种哺乳动物细胞系染色体制片条件优化研究

Optimization of Chromosome Production Conditions for Six Mammalian Cell Lines

为提高胞核学检验的检查效率,采用6种具有种属代表性的常用传代细胞系对影响检验的关键步骤(染色体制片条件)进行优化,并对这六种传代细胞系的染色体数目进行了统计。实验以CHO、BHK-21、ST、OA3.Ts、MDBK、RK-13六种疫苗生产中常用的传代细胞系作为材料,对影响染色体制片效果几个主要条件(秋水仙素浓度、KCl浓度及低渗处理时间)进行了优化,并对这六种细胞染色体数目分布进行了检查。结果发现,在细胞用25 cm^2细胞培养瓶1∶2比例传代24~48 h时,秋水仙素浓度为1μg/mL,处理60 min后处在中期分裂相的细胞最多,视野中染色体长度适中;用0.4%KCl溶液低渗处理40 min,细胞中染色体在载玻片上形态最佳,分散度最好。优化后的染色体制片条件,可适用于多种细胞的染色体制片,结果重复性好,染色体形态和分散度均满足胞核学检验要求,可为胞核学检验的流程标准化制定提供有益参考。

In order to improve the efficiency of karyotype analysis,different chromosome conditions were compared to optimize this key step affecting whole process by employing six common cell lines, and the number of chromosomes in these six cell lines was counted. Six mammalian cell lines CHO, BHK-21, ST, OA3.Ts, MDBK and RK-13 were used to explore the optimized terms such as colchicine concentration, KCl solution concentration and low osmotic treatment time, which play major roles in chromosome slicing. The distribution of chromosomes was also observed. The results showed that the number of metaphase cells reached up to the most and chromosome lengths were moderate when cells were sub-cultured in 25 cm^2 cell culture flask at a ratio of 1∶2 for 24~48 hours, with being treated by colchicine for 60 min at the concentration of 1 μg/mL. Chromosome morphology and the diapersity both presented the best when treated with 0.4% KCl solution for 40 minutes. With a good repeatability, the optimized conditions of chromosome slicing preparation could be applied to many kinds of cells, of which chromosome morphology and dispersion meet the requirements of karyotype analysis. This study provides a useful reference for the procedure standardization of the karyotype analysis.