饲养层密度对兔类ES细胞分离培养的影响

摘要

探讨饲养层细胞接种密度对兔类ES细胞分离培养影响,筛选适合兔类ES细胞分离培养最适饲养层密度.将兔囊胚分别接种到密度为10×103、20×103和40×103个·cm-2的小鼠胚胎成纤维细胞(Mouse embryonic fibroblast cell,MEF)饲养层上,观察兔类ES细胞贴壁和克隆生长情况,并对所得兔类ES细胞进行碱性磷酸酶和Oct-4检测.结果表明,接种于密度为20×103个·cm-2饲养层上的囊胚贴壁率(82.5％)、内细胞团(Inner cell mass,ICM)形成率(67.5％)高于接种于密度为40×103个·cm-2的饲养层上囊胚贴壁率(80.0％)、内细胞团(Inner cell mass,ICM)形成率(65.7％),差异不显著(P＞0.05)；显著高于(P＜0.05)接种于密度为10×103个·cm-2饲养层上的囊胚贴壁率(55.6％)、ICM形成率(44.4％).密度为20×103个·cm-2饲养层上的兔类ES细胞F1、F2代细胞克隆率均显著高于其他两组(P＜0.05),最高已传至20代；获得的兔类ES细胞经碱性磷酸酶染色及表面抗原Oct-4表达检测均呈阳性.密度为20×103个·cm-2的MEF饲养层可更好支持兔类ES细胞体外培养.%To explore the effects of different densities of feeder layer cells on isolation and culturing of rabbit ES-like cells and select the better culture system.Rabbit blastulas were cultured at different densities(10 × 103,20 × 103,and 40 × 103 · cm-2) of mouse embryonic fibroblast cell (MEF) as feeder layer,rabbit ES-like cell adherence and growth behavior were observed and express pluripotency markers were detected,such as AKP and Oct-4.The results showed that the attachment rate of blastulas and ICM formation rate at a density of 20 × 103 · cm-2 feeder layer were higher than those at 40×103 · cm-2 (82.5％ vs 80.0％; 67.5％ vs 65.7％,respectively),but the two groups were not significantly different (P＞0.05).While stem cells cultured at a density of 20 × 103 · cm-2 feeder layer were significantly higher than at 10 × 103 · cm-2 feeder layer (82.5％ vs 55.6％; 67.5％ vs 44.4％,respectively) (P＜0.05).The cell clone rate of F1、F2 generation of rabbit ES-like cells at a density of 20 × 103 · cm-2 feeder layer was significantly higher than other two groups (P＜0.05),and the stem cells obtained were up to the 20th passages and expression of AKP and Oct-4 were positive.In conclusion,the MEF feeder layer at a density of 20 × 103 · cm-2 may be better suitable for rabbit ES-like cells culture in vitro.