摘要
取8周后的雌性昆明小鼠进行超排,取卵母细胞用作核受体,收集卵母细胞周围的卵丘细胞作核供体,进行体细胞核移植。核移植重构胚经SrCl2激活处理6h后,与改良的M16培养液和小鼠输卵管上皮细胞共培养;将发育到早期囊胚阶段的重构胚转移至小鼠胎儿成纤维细胞饲养层上,添加含心肌细胞培养液的ES细胞培养液;把孵出的ICM进行消化接种培养,对孵出的ES细胞集落进行鉴定培养。结果显示,以小鼠卵丘细胞为核供体,体细胞核移植重构胚激活率为65.23%,囊胚发育率为11.69%;9个核移植重构囊胚中分离出ES细胞集落,分离率为2.77%;分离出的核移植ES细胞集落具有岛屿状团状隆起结构、碱性磷酸酶染色呈阳性,体外分化可形成类胚体,并能分化成上皮样或梭形细胞。ES细胞集落经常规冻存和复苏后,显示出同冻存前相似的集落形态,并具有较强的增殖能力。实验证实小鼠输卵管上皮细胞、改良的M16培养液及含心肌细胞培养液的ES细胞培养液可以更为成功地运用于小鼠的体细胞核移植及ES细胞的分离培养研究。
Female mice after 8 weeks were superovulated with PMSG and hCG, and their eggs were taken as recipients. Meanwhile cumulus cells around oocytes were also collected and used as donor cells in somatic cell nuclear transfer. Oocytes injected with donor cell nuclei were activated for 6 h by treatment in M16 media modified containing SrCl_2, and then cocultured with mouse oviduct epithelial cell in mM16 medium. When being in the stage of blastocyst, they were transfered on the feeder layers of mouse embryonic fibroblasts, adding ES cell media conditioned. After hatched from blastocysts, ICM were isolated and trypsinized, and then cocultured continuously to gain ES cell masses. Results indicated that activation rate of embryos reconstructed was 65.23%, development rate of blastocyst was 11.69%; ES cell colonies were isolated from 9 blastocysts reconstructed, isolation rate was 2.77%. ES cell colonies isolated were with island-like images and strong positive by AKP staining, could become embryo bodies and spontaneously differentiate into epidermal-like cells around them in vitro. In addition, after frozen and thawed routinely, ES cell colonies were with strong proliferation and previous image. It indicates that mouse oviduct epithelial cell, modified M16 media and ES cell media with cardiomyocyte media can be more successfully applied in mouse somatic cell nuclear transfer and isolation of ES cell from reconstructed embryos.
