中国农业科学2012，Vol.45Issue(12)：2474-2481,8.DOI:10.3864/i.issn.0578-1752.2012.12.017
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
Bovine Embryonic Stem Cells Culture on Mixed Feeder Layers with Mouse and Bovine Embryonic Fibroblasts in Vitro
摘要
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 I : 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 fibrobiasts, 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.关键词
牛/胚胎干细胞/成纤维细胞/混合饲养层Key words
bovine/ ebryonic stem cell/ ebryonic fibrobiasts/ mixed feeder layer引用本文复制引用
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):2474-2481,8.基金项目
国家“863”计划项目(2008AA101005) （2008AA101005）
