山羊胚胎干细胞的分离与培养

Isolation and Cultivation of Goat Embryo Stem Cells

摘要:

对关中奶山羊配种后6~7天的桑椹胚和囊胚, 分别采用全胚培养法、酶消化法和免疫外科法进行处理。将处理后的胚胎培养于小鼠胎儿成纤维细胞(MEF)饲养层上, 分离培养山羊胚胎干细胞(Embryonic stem cell, ESC)。对分离传代的山羊ESCs分别进行免疫组化染色, RT-PCR检测和体外诱导分化试验。结果表明, 全胚培养法易于胚胎贴壁形成原代集落, 采用全胚培养法获得的ESCs有一株目前已传至18代。山羊ESCs Nanong、Oct4、SSEA-3免疫组化染色呈阳性, SSEA-1免疫组化染色呈弱阳性, SSEA-4免疫组化染色呈阴性, RT-PCR检测显示其表达Nanog、Oct4、端粒酶、CD117。山羊ESCs经DMSO体外诱导可以向心肌细胞分化, 这些试验均表明该细胞具有ESCs的生物学特性。

Abstract:

Morulaes and blastocysts obtained from Guanzhong dairy goats 6~7 days after mating were treated with whole embryo cultivaton, enzymatic digestion and immunosurgery separately. The goat embryonic stem cells (ESC) were isolated and cultured on a feeder layer of mitomycin-inactivated mouse embryo fibroblasts (MEF). The characteristics of goat ESCs were analyzed by immunohistochemisty, RT-PCR and inducing differentiation in vitro. The results indicated that the embryos were easier to attach the culture dish and form primary colonies with whole embryo method. There were colonies that maintained undifferentiated for 18 passages. The ESCs expressed the protein of Nanog, Oct4 and SSEA-3, whereas the protein of SSEA-4 was absent and the protein of SSEA-1 was weakly expressed. In addition, the genes of Nanog, Oct4, TERT and CD117 were expressed in goat ESCs. The cells also could differentiate to myocardial cells when induced in vitro by DMSO. These results suggest that the goat ESCs have characteristics of ESCs.