鸡胚胎干细胞饲养层培养体系的建立  

Establishment of the Feeder Layer Optimal Culture System for Chicken Embryonic Stem Cells

作　　者：邹清雁[1] 张淑莲[1] 郑曲波[1] 孔祥平[1] 肖荣 张宜俊[1] 

机构地区：[1]解放军第458医院解放军传染病防治中心,广州510602

出　　处：《上海实验动物科学》2000年第4期206-209,共4页Shanghai Laboratory Animal Science

摘　　要：采用孵化 8～ 1 2 d鸡胚 ,用胰酶消化 ,以含 1 0 % NBS的 DMEM溶液为培养液 ,分离获得鸡胚成纤维细胞 (CEF) ,建立了培养鸡 ES细胞的饲养层细胞。经过 CEF酶活力法筛选 ,建立了 ES细胞的条件培养基 ,其组成配方为 1 0 % NBS、5 % FBS、 2 .5 %的鸡胚提取物 ,1× 1 0 6/ L m L IF,1 0μg/ L人 b FGF,40μg/ L伴白蛋白 ,0 .1 4mmolβ-ME的高糖DMEM。鸡类 ES细胞 (AKP阳性细胞 ,因未经遗传学和免疫组织化学鉴定 ,故称类 ES细胞 )在经丝裂霉素 -C处理的饲养层细胞上和条件培养基中可传至 9代仍保持良好的增殖能力和未分化状态。The chicken embryos incubated for 8~12 days were trypsinized to dissociate the chicken embryonic fibroblasts(CEF). CEF were maintained in DMEM containing 4.5 g/L glucose, 10% newborn calf serum(NBS) as the feeder layer for culturing chicken ES cells after treated with 10 mg/L Mitomycin C. In order to define the growth requirement for proliferation of ES cells, the sera and the chicken embryonic extract were screened by the methods of testing CEF's dehydrogenase activity, and the chicken ES cell's optimal culturing system which composed of DMEM(high glucose) containing 10%NCS, 5%FBS, 2.5% chicken embryo extract, 10μg/L human bFGF, 1×10 6/L mLIF, 40μg/L Conalbumin, and 0.14mmol β Mercaptoethanol(β ME) were established. The alkaline phosphatase positive(chicken ES ) cells growing well and maintained undifferentiated until 9 passages in the feeder layers of the optimal culturing system.

关 键 词：鸡 饲养层 鸡胚成纤维细胞 胚胎干细胞 

分 类 号：Q813[生物学—生物工程]