昆明小鼠3.5d和4d囊胚不同分离方法的比较

背景：远交系的昆明小鼠作为中国自然科学研究主要实验动物,其胚胎干细胞建系的成功率一直很低.目的：探讨昆明小鼠胚胎干细胞体外分离培养的最佳方法和最适合的采胚时间.方法：采集孕3.5 d和4 d的囊胚分别以免疫外科法和全胚培养法在小鼠胚胎成纤维细胞饲养层上分离和克隆昆明小鼠胚胎干细胞集落.结果与结论：免疫外科法和全胚法培养3.5 d囊胚的内细胞团贴壁率和原代克隆形成率差异均不显著（P 〉 0.05）;全胚法培养4 d囊胚的内细胞团贴壁率显著高于免疫外科法（P 〈 0.05）,但原代克隆形成率显著低于免疫外科法（P 〈 0.05）;全胚法培养4 d囊胚的原代克隆形成率显著高于3.5 d囊胚（P 〈 0.05）;免疫外科法分离4 d囊胚内细胞团的贴壁率和原代克隆形成率显著高于以同样方法分离培养的3.5 d囊胚（P 〈 0.05）.结果显示用免疫外科法分离4 d囊胚更适合于昆明小鼠胚胎干细胞的体外分离培养.

Comparison of different methods to isolate 3.5-day and 4-day blastocysts from Kunming mice

BACKGROUND： Outbreed Kunming mice as the main experimental animals for natural science research in China have a low successful rate in establishment of embryonic stem cell line. OBJECTIVE： To explore the method of isolation and culture of murine embryonic stem cells and the best time to collect embryos of Kunming species. METHODS： Blastocysts obtained from 3.5-day and 4-day embryos after mating were treated with intact embryo cultivation and immuosurgery. Inner cell masses were isolated and cultured on a feeder layer of mouse embryo fobroblasts （MEF）. And colony growth was observed. RESULTS AND CONCLUSION： Adherent rate of inner cell mass by inatact embryo cultural method were higher than those by immunosurgical method from 4-day （P 0.05）, but in the rate of primary colony formation, the latter was better than the former. And, in the rate of primary colony formation, 4-day was better than 3.5-day by intact embryo culture method （P 0.05）. By immunosurgical method, adherent rate of inner cell mass and the rate of primary colony formation from 4-day were higer than those of 3.5-day （P 0.05）. Immunosurgical method to isolate 4-day embryos is helpful to culture embryonic stem cells of Kunming mice in vitro.