蒙古绵羊体外受精胚胎干细胞建系培养研究  

Establishment of cultivation system for embryonic stem cell lines of in vitro fertilization embryos in Mongolia sheep

作　　者：靳木子[1,2] 吴慧[1] 邰大鹏[1] 杨文亮[1] 仓明[1] 刘东军[1] 

机构地区：[1]内蒙古大学,教育部哺乳动物生殖生物学及生物技术重点实验室,内蒙古呼和浩特010021 [2]内蒙古国际旅行卫生保健中心,内蒙古呼和浩特010020

出　　处：《畜牧与兽医》2015年第11期28-33,共6页Animal Husbandry & Veterinary Medicine

基　　金：转基因生物新品种培育重大专项(2011ZX08008-002)

摘　　要：蒙古绵羊体外受精的胚胎干细胞(SESC)一直未能成功建系,确定一种适合的培养体系及传代方法对其成功建系有着重要的意义。本研究将获得的体外受精胚胎直接培养,以胎鼠成纤维细胞为饲养层,运用机械传代法传代,采用培养液Ⅰ和培养液Ⅱ两种不同的培养液培养绵羊胚胎干细胞。采用形态鉴定、碱性磷酸酶染色、类胚体形成,免疫荧光染色等方法对获得的SESC进行干细胞检测。结果显示:培养液Ⅰ和培养液Ⅱ都可用于SESC的培养,而无血清的培养液Ⅱ更利于细胞的生长;机械传代法适合于SESC的传代培养;冷冻复苏之后经过形态鉴定、碱性磷酸酶染色、体外分化以及免疫荧光染色,均与胚胎干细胞的形态特征一致。Embryonic stem cells （SESC） of in vitro fertilization embryos in Mongolian sheep have not been founded successfully to date, a suitable culture system and passage method in founding SESC is immediately required. In this study, in vivo fertilized embryos were then directly cultured in serum-containing medium I or 11. During the cultivation, mouse fetal fibroblasts were selected as the feeder layer and mechanical method was used to passage SESC. Subsequently, we identified the SESC by morphology, alkaline phosphatase staining, the formation of embryoid body and immunofluorescence staining which are typical characteristics of ESCs. The result showed that both kinds of media can be used for the cultivation of the SESC, but the serum-free medium with definite components was more favourable for SESC growth. The mechanical method was suitable to the passage of SESC. There was no change before and after cryopreservation and thawing with regard to AgESC morphology, alkaline phosphatase staining and formation of embryoid bodies or immunofluorescence staining.

关 键 词：绵羊 体外受精 胚胎干细胞 建系 

分 类 号：Q2-33[生物学—细胞生物学]