山羊类ES细胞的分离与克隆

Isolation and Cloning of ES-like Cells of Goat

采集山羊交配后 6～ 8d的桑椹胚、囊胚和孵化囊胚 ,将桑椹胚和囊胚分别放在小鼠原代胎儿成纤维细胞(PMEF)饲养层和同源原代胎儿成纤维细胞 (PGEF)饲养层上比较其脱带时间及脱带率。脱带后 ,将各自一半胚胎切割 ,把含 ICM的半胚分别放在相应饲养层上进行培养 ,另一半整胚在各自饲养层上继续培养 ,而孵化囊胚直接于PGEF饲养层上培养。当 ICM增殖一定程度时进行传代 ,以比较其类 ES细胞分离与克隆的效果。结果表明 ,在 2种不同饲养层上 ,囊胚的脱带时间均短于桑椹胚 ,囊胚的脱带率均高于桑椹胚 ,而饲养层的种类对胚胎的脱带时间以及脱带率影响不大。脱带切割囊胚不论在 PMEF还是在 PGEF饲养层上 ,其贴壁时间均短于脱带整胚及孵化囊胚 ,而贴壁率高于脱带整胚 ,与孵化囊胚相似。脱带整胚及脱带切割胚在 PMEF饲养层上所获类 ES细胞只能维持 3代 ,而在PGEF饲养层上 ,脱带切割半胚和孵化囊胚所获类 ES细胞传至 5代。由此认为 ,对脱带后的胚胎进行切割处理 ,有利于 ICM的贴壁和增殖 ;应用同源原代胎儿成纤维细胞饲养层培养系统 ,有利于类

Morulae,blastocysts and hatched blastocysts were collected from the uterine horns of local goat 6 8 d after mating.The morulae and blastocysts were cultured on the primary mouse embryonic fibroblasts(PMEF) feeder and primary homogenous(goat) embryonic fibroblasts(PGEF) feeder to compare their hatching time and hatching rate from the zone pellucida.After hatching,half of the blastocysts were bisected and the bisected embryos with inner cell mass(ICM) were cultured on the feeder as before.The other halves were continued to culture on the feeder as before.The hatched blastocysts collected from uterine horns were directly cultured on the PGEF feeder.After the ICM proliferated to some extent,the clones were passaged to compare the effects of isolation and cloning of embryonic stem like(ES like) cells.The results indicated that the time of hatching from the zone pellucida of blastocysts was shorter than that of morulae,and the rates of hatching were higher on the two types of feeder.There was no difference in effects of different types of feeders on the time and rate of hatching.The attachment time of bisected embryos with ICM was shorter than that of intact blastocysts and hatched blastocysts and the attachment rate of bisected embryos was higher than that of intact blastocysts but was as high as that of hatched blastocysts.The third passages of ES like cells were obtained from the intact blastocysts and bisected embryos on the PMEF feeder and the fifth passages of ES like cells were obtained on the PGEF feeder.In conclusion,the research shows that the bisecton of embryo after hatching is beneficial to the attachment and proliferation of ICM and the culture system of primary homogenous embryonic fibroblast feeder is beneficial to the isolation and cloning of ES like cells.