摘要
目的 建立有效的胚胎成纤维细胞饲养层体系 ,用于分离和克隆胚胎干细胞研究。方法 取胎鼠组织制备小鼠原代成纤维细胞 ,观察不同分离和培养条件对细胞增殖的影响 ,以及丝裂霉素C对细胞增殖的抑制作用 ;细胞增殖的数量用MTT法测定。结果 分离小鼠胚胎原代成纤维细胞的最适胎龄是 12 5～ 14 5d ;2 0℃～ 2 5℃时 ,用 0 2 5 %胰蛋白酶消化组织的最佳时间为 3min ;可在培养 3～ 6d后进行传代。细胞冻存于 - 80℃ 3～ 4个月 ,复苏后能正常传代。丝裂霉素C抑制胚胎成纤维细胞增殖的合适浓度为每毫升 2 0 μg 10 5细胞 ,作用 2～ 4h ,可维持小鼠原代胚胎成纤维细胞 9d内不增殖也不死亡。结论 在合适条件下 ,小鼠原代胚胎成纤维细胞能顺利分离和培养 ,并能多次传代和冻存 ;经丝裂霉素C处理后增殖受抑制 ,可作为饲养层细胞用于胚胎干细胞的研究。
Objective To establish mouse fibroblast cells as feeder cells in order to isolate and clone mouse embryonic stem cells in vitro.Methods The mouse primary embryonic fibroblasts were isolated from mouse fetus of different gestational ages,under different separation and culture conditions.The proliferation of fibroblasts and inhibition of mytomycin C were observed by measuring cell numbers using MTT assay.Results The optimal gestational age for isolation of mouse embryonic fibroblasts was from on 12.5 to 14.5 day.At the temperature of 20 to 25℃,the optimal time for digestion of fetus tissue to obtain fibroblasts was 3 min with digestion solution containing 0.25% of trypsin.The optimal concentration of mytomycin c to inhibit proliferation of fibroblasts was 20μg/10 5 cells for 2 to 4 hours,at that condition of treatment of mytomycin C the fibroblasts were alive for 9 days without proliferation.Conclusion In this study the mouse embryonic fibroblasts were successfully established for growth of mouse embryonic stem cells.
出处
《中国实验动物学杂志》
2002年第4期215-219,共5页
Chinese Journal of Laboratory Animal Science
