摘要
取成年小鼠唇部皮肤进行培养, 分离成纤维细胞并血清饥饿培养1 周, 用作核供体。对成年小鼠进行超排, 取卵母细胞用作核受体,核移植重构胚经SrCl2 激活处理6 h后, 同mM16培养液和小鼠输卵管上皮细胞共培养, 把发育到早期囊胚的重构胚转移至小鼠胎儿成纤维细胞饲养层上, 添加ES细胞条件培养液,消化分离ICM,然后接种培养, 对孵出的ES细胞样集落进行鉴定培养。结果显示, 小鼠唇部皮肤成纤维细胞为核供体, 核移植重构胚2 细胞率为54 05%, 桑椹胚率17 14%, 囊胚率6 90 %, 对照组卵丘细胞的核移植重构胚2 细胞率为60 00%, 桑椹胚率21 85%, 囊胚率11 69 %, 但2种供体细胞在支持核移植重构胚发育能力上差异不显著。成纤维细胞重构囊胚中6个囊胚分离出ES细胞样集落, 3个ES细胞样集落可稳定传代; 对照组卵丘细胞重构囊胚中9个囊胚中分离出ES细胞样集落, 5个ES细胞样集落可稳定传代。从核移植重构胚中分离出的ES细胞样集落具有岛状或巢状群体生长形态,生长旺盛的集落可自发分化成单个散在或片状存在的上皮样或梭形细胞, 碱性磷酸酶检测为阳性, 常规冻存复苏, 仍显示ES细胞特征。
Fibroblasts were isolated from adult mouse lip skin and cultured,and then starved 1 week with 0.5% FBS media before used as donor cells. In addition, adult female mice were superovulated with PMSG and hCG, and their eggs were taken as recipients. Embryos reconstructed were activated 6 hours with 10 mmol/L SrCl_2 2 hours after injected nucleus, and then cocultured with mouse oviduct epidermal cells, adding M16 media modified. When being in the stage of blastocyst, they were transfered on the feeder layers of mouse embryonic fibroblasts, adding rat cardiomyocyte media conditioned. After hatched from blastocysts, ICM were isolated and trypsinized, and then co-cultured continuously to gain ES cell masses. Results indicated that 2-cell rate of embryos reconstructed with fibroblasts was 54.05%, development rate of morula was 17.14%, blastocyst rate was 6.90%. Under the same circumstance, 2-cell rate of embryos reconstructed with cumulus cells was 60.00%, development rate of morula was 21.85%, blastocyst rate was 11.69 %, but developmental capacity of embryo reconstructed was no difference between two donor cells. ES cell -like colonies were isolated from 6 blastocysts with fibroblasts, in which 3 ES cell-like colonies could be passaged and cultured successfully. In control group ES cell -like colonies were harvested from 9 blastocysts, in which 5 ES cell-like colonies could be passaged successfully. ES cells isolated were island-like, positive by AKP staining, and could spontaneously differentiate into epidermal or shuttle-like cells around them in vitro. After routinely frozen and thawed, they were with characteristics of ES cell.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2005年第6期559-563,共5页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家"863"高技术项目(2001AA213081)
