The aim of this study was to optimize and set up better goat feeder layer cell, culture medium, cell factors and passaging method for better growth of goat ES-like cells. Blastocysts obtained by whole embryo cultured, trypsin digestion and mechanical dissociation after enzymatic treatment from Wuhan goats 6-7 days after mating were cultivated. The effect of different cell layers, different cell factors in culture medium and different passaging methods on the cell survival and differentiation were compared. Alkaline phosphataseassay (ALP) and immunohistochemi-cal staining method were used to detect the activity of AKT and the expression of ES cells specific transcription factor including SSEA-1 and Oct-4, and further study the differentiation ability of goat ES-like cells. Significant differences are not noted between cell types that C2C12 can promote adherence(100%), better than MEF(81. 8%)and GEF(88. 5%), but not benefit for restraining undifferentiation state; the division of inner cell mass with high concentration of LIF and SCF cell factors could help the cells passage in undifferential condition (8 passages) ; the media contai-ning LIF and SCF, while adding heparin and insulin, was more conducive to cells passage (9 passages) ; the ESCs expressed the protein of SSEA-1 and Oct-4, in vitro these cells could differentiate into epithelial-like cells, vacuole-like cells and cardiomyocytes. The goat ES-like cells differentiating into variety cell types were obtained successfully.%旨在优化并建立有利于山羊类ES细胞生长的饲养层细胞、培养基、细胞因子和传代方法.本研究选择武汉市本地白山羊配种6～7 d后的胚胎,通过全胚培养法、酶消化与机械分离结合法分离山羊类ES细胞,并在不同的细胞饲养层中饲养、在培养基中添加不同的生长因子、采用不同的传代方法,比较不同培养条件对山羊类ES细胞生长和增殖的影响.通过碱性磷酸酶染色和免疫组化染色法鉴定ES细胞特异生物标记AKT、SSEA-1和Oct-4的表达,并将得到的山羊类ES细胞进行体外分化.结果表明,与小鼠胎儿成纤维细胞(Mouse embryonic fibroblast,MEF)和山羊胎儿成纤维细胞(Goat embryonic fibroblast,GEF)相比,C2C12更有利于细胞的贴壁,但差异不显著(P＞0.05)；细胞传代时,在高浓度细胞因子LIF(Leukemia inhibitory factor)和SCF(Stem cell factor)中处理ICM(Inner cell mass),更有利于细胞传代(传至第8代)；培养基中添加LIF和SCF的同时,添加肝素和胰岛素,更有利于细胞的传代(传至第9代)；山羊类ES细胞中SSEA-1(Stage-specific embryonic antigens-1)和Oct-4免疫组化染色呈阳性,在体外培养时可以分化为上皮样细胞、空泡样细胞和跳动的类心肌细胞.成功分离得到能自然分化成不同类型细胞的山羊类ES细胞.
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