牛和小鼠胎儿成纤维细胞混合制成的饲养层对牛胚胎干细胞的体外培养

doi:10.3864/j.issn.0578-1752.2012.12.017

摘要/Abstract

摘要： 【目的】建立适合的牛胚胎干细胞培养环境，保持其未分化状态，保证细胞的全能性。【方法】分别培养小鼠胎儿成纤维细胞（mouse embryonic fibroblast，MEF）和牛胎儿成纤维细胞（bovine embryonic fibroblast，BEF），用丝裂霉素C处理两种成纤维细胞分别计数后按1﹕0、1﹕1、2﹕1、1﹕2和0﹕1的比例制成混合饲养层，观察牛胚胎干细胞的生长状态，并对生长在混合饲养层上的牛胚胎干细胞进行碱性磷酸酶，OCT4和SSEA-1免疫组化检测，OCT4、SOX2、NANOG mRNA 表达RT-PCR检测、体外分化形成拟胚体等试验。【结果】①生长在混合饲养层上的牛胚胎干细胞比单独生长在小鼠或牛胎儿成纤维细胞饲养层的结构致密、与滋养层界限明显；②小鼠胎儿成纤维细胞和牛胎儿成纤维细胞在1﹕1比例混合下牛胚胎干细胞克隆结构致密、显著堆积且边缘轮廓清晰优于其它比例组合；③获得的牛胚胎干细胞AKP染色及OCT4、SSEA-1抗原表达均成阳性，分别在体外分化培养形成了拟胚体。【结论】小鼠和牛胎儿成纤维细胞在以1﹕1的比例混合下制成的饲养层可更好地支持牛胚胎干细胞的体外培养，获得的克隆形态最好。

关键词: 牛, 胚胎干细胞, 成纤维细胞, 混合饲养层

Abstract: 【Objective】The aim of this study was to establish a suitable culture environment for bovine embryo stem cell.【Method】Bovine and mouse fibroblast cells were treated for 2.0-3.0 h with mitomycin C, respectively. Mixed feeder layer was that cells which were mixed according to 1﹕0, 1﹕1, 1﹕2, 2﹕1, 0﹕1 were cultured in the center plate coated by gelatin at 2-2.5×104 per well. Growth states of bovine embryonic stem cells were observed in different feeder layers and undifferentiated phenotypes were detected, including expression of alkaline phosphatase, and presence of OCT-4, SOX2, NANOG and cell marker OCT-4, SSEA-1. Without feeder layer, bovine embryonic stem cell differentiation was observed in vitro.【Result】Comparison of bovine embryonic stem cell growth state on different feeder layers, the clonal morphology on the mixed feeder layer was significantly better than that on the mouse embryonic fibroblast feeder layer or bovine embryonic fibroblast feeder layer. When mouse embryonic fibroblasts and bovine embryonic fibroblasts were mixed at a ratio of 1﹕1 bovine embryonic stem cells accumulated significantly and cloning edge was clear, obvious and full. Expression of alkaline phosphatase, antigen of OCT-4 and SSEA-1 were strongly positive. Specific bands of OCT-4, SOX2 and NANOG mRNA appeared, respectively. Bovine embryonic stem cells on mixed feeder layer was able to form embryoid bodies.【Conclusion】Compared with conventional feeder layers prepared by mouse embryonic fibroblasts or bovine embryonic fibroblasts, the mixed cells feeder layer mixed at a ratio of 1﹕1 may be better suitable for bovine embryonic stem cell culture in vitro and obtain better bovine rembryonic stem cell clonal morphology.

Key words: bovine, ebryonic stem cell, ebryonic fibroblasts, mixed feeder layer

丛姗, 王瑞, 温建勋, 郝斐, 李硕, 梁浩, 刘东军. 牛和小鼠胎儿成纤维细胞混合制成的饲养层对牛胚胎干细胞的体外培养[J]. 中国农业科学, 2012, 45(12): 2474-2481.

CONG Shan, WANG Rui, WEN Jian-Xun, HAO Fei, LI Shuo, LIANG Hao, LIU Dong-Jun. Bovine Embryonic Stem Cells Culture on Mixed Feeder Layers with Mouse and Bovine Embryonic Fibroblasts in Vitro[J]. Scientia Agricultura Sinica, 2012, 45(12): 2474-2481.

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