饲养层密度对兔类ES细胞分离培养的影响

Effect of Densities of Feeder Layers for Isolation and Culturing of Rabbit ES-like Cells

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 embryonicfibroblast 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 cellmass,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(Innercell 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 cultu- ring of rabbit ES-like cells and select the better culture system. Rabbit blastulas were cultured at different densities（10×10^3 , 20 × 10^3 , and 40× 10^3 ·cm-2 ） of mouse embryonic fibroblast cell （MEF） as feeder layer, rabbit ES-like cell adherence and growth behavior were observed and ex- press 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× 10^3 · cm-2 feeder layer were higher than those at 40×10^3 ·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）〈 10^3 ·cm-2feeder layer were significantly higher than at 10×10^3 ·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 ceils at a density of 20 × 10^3 ·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 ×10^3 ·cm-2may be better suitable for rabbit ES-like cells culture in vitro.