无血清无饲养层培养体系在绵羊胚胎干细胞分离中的应用

目的：本文采用N2B27无血清无饲养层的完全已知成份的培养体系，通过机械分离法分离不同阶段的绵羊囊胚，观察不同阶段囊胚对分离绵羊类胚胎干细胞的影响。方法：本实验采用N2B27无血清无饲养层成份完全已知的培养体系，利用机械分离法对不同阶段的绵羊囊胚进行分离，观察其绵羊类胚胎干细胞的原代集落形成率，以及AKP 染色，多潜能性候选基因Oct-4 和Sox-2 免疫荧光检测。结果：分离早期阶段绵羊囊胚获得的绵羊类胚胎干细胞的形成率显著低于扩张（孵化）阶段囊胚（19.6%（11/56） vs 36.9%（31/84））（P ＜ 0.05），同时早期和扩张（孵化）阶段绵羊囊胚的AKP 染色和多潜能性候选基因Oct-4、Sox-2 的表达呈阳性。结论：N2B27无血清无饲养层培养体系是一种有效分离绵羊类胚胎干细胞的培养基，同时分离绵羊扩张（孵化）阶段的囊胚可以显著的提高原代类胚胎干细胞的建系率，为提高绵羊类胚胎干细胞的建系奠定了基础。

The Application of the Separation of Sheep Embryonic Stem Cells in Serum-and Feeder-free Conditions

Objective： To observe the influence of the separation of sheep embryonic stem cells in vitro culture blastocysts at different developmental stages by N2B27 serum- and feeder-flee conditions. Methods： Mechanical separation was used to isolate the in vitro culture blastocysts at different developmental stages by N2B27 serum- and feeder-free conditions, and then we observed the formation rates of primary oESC-like, AKP dying, expression of multipotent candidate genes Oct-4 and Sox-2. Results： The formation rates of oESC-like of early blastocysts was lower extended blastocysts （19.6% （11/56） vs 36.9%（31/84））（P 〈0.05）. It was all positive for the assays of alkaline phosphatase and expression of multipotent candidate genes Oct-4 and Sox-2. Conclusion： This is an effective method that the separation of sheep embryonic stem cells in N2B27 serum- and feeder-flee conditions. The extended blastocysts can improve the formation rates of primary oESC-like.