【作者】 刘羿羿；

【导师】 周欢敏；

【作者基本信息】 内蒙古农业大学 ， 动物发育生物学与生物技术， 2010， 博士

【摘要】 为摸索山羊胚胎干细胞体外培养条件,本实验从胚胎来源、接种方法、饲养层、包被条件、生长因子等方面对影响山羊胚胎干细胞的分离、培养的因素进行了研究,并探索了山羊PGCs的体外培养条件,最后对胚胎干细胞和PGCs进行了鉴定。1.通过超数排卵、体外受精、核移植方法获取囊胚。将体内获取的山羊囊胚切半或将全胚接种到山羊胎儿成纤维细胞饲养层、胎牛肝脏成纤维细胞饲养层和小鼠胚胎原代成纤维细胞饲养层细胞上,比较贴壁率和传代次数后发现饲养层细胞和胚胎处理方法对山羊ICM贴壁和传代培养影响均显著,切半的山羊体内发育的囊胚在小鼠胚胎原代成纤维细胞上贴壁率最高为55.6%,可以在体外传至6代,而在山羊胎儿成纤维细胞饲养层上孵化的全胚贴壁率最低为10%,体外仅传2代。2.分别采用高糖DMEM及低糖DMEM/F12干细胞培养液在小鼠胚胎原代饲养层细胞上培养切半的体外发育囊胚,待囊胚贴壁后用玻璃针剥离内细胞团,酶消化后传代培养。结果表明:低糖DMEM/F12更适宜于山羊ICM的增殖与克隆形成,ICM在低糖培养液中贴壁率为35.7%,可传5代,在高糖培养液中贴壁率为26.3%,可传3代,差异显著。3.为比较明胶和ECM对山羊类胚胎干细胞贴壁和培养的影响,将切半的体外发育的胚胎接种到小鼠胎儿原代成纤维细胞饲养层上,用低糖培养液培养,结果发现在明胶包被的培养皿上ICM贴壁率为35.7%,在ECM包被的培养皿上ICM贴壁率稍高,为40%,均可传5代,差异不显著。本实验比较了体外发育的囊胚和克隆囊胚的贴壁率并传代培养,结果发现:在ECM包被的培养皿、小鼠胚胎原代成纤维细胞饲养层上,低糖培养的条件下,核重编程囊胚ICM的贴壁率为25%,最高传2代,体外发育的囊胚ICM贴壁率为40%,最高传5代,差异极显著。4.将内蒙古白绒山羊胎儿的原始生殖细胞(PGCs)与生殖嵴周围细胞共同分离,在三种培养体系中传代培养,结果表明:未添加任何因子的A培养体系仅能传1代,仅添加LIF及Insulin的B培养体系可以传至4代,添加LIF,Insulin及优质胎牛血清DMEM/F12的C组培养体系可以传至9代。5.对所分离的山羊类胚胎干细胞和PGCs细胞进行形态学分析、RT-PCR鉴定、AKP染色及免疫荧光检测,结果发现:山羊类胚胎干细胞和PGCs细胞可以形成类似鸟巢状的集落,细胞界限不清,核较大,易于周围饲养层细胞区别,AKP鉴定为阳性,RT-PCR检测β-actin、hTERT、GAPDH、Nanog、Oct3/4为阳性,免疫荧光鉴定发现Oct3/4、SSEA-3、SSEA-4呈阳性,在体外可以形成有空腔的类胚体,可以体外分化为神经样细胞,脂肪细胞和上皮细胞状细胞。更多还原

【Abstract】 Experiments were performed to determine the effect factor of isolation , cloning and passage of embryonic stem cells ( ES) in goat , factors include sourse of embryos , plant methed ,feeder cells ,coat conditions and growth factors.Then culture and identified the goat PGCs cells in vitro .1.According to ICM goat embryos in vivo culture conditions.plant half blastocyst on MEF feeder culture in high glucose and low glucose DMEM DMEM/F12 medium .After the blastocyst attached stripping ICM by glass needle. The results showed: low glucose DMEM more suitable for goats ICM proliferation and colony formation ,in the culture medium 1 ICM adherence rate was 35.7% can be passaged 5 times, the culture medium 2 adherence rate was 26.3% can be passaged 2 times,the difference was significant. Planted half or hatching goat blastocyst on goat fetal fibroblast feeder layer, bovine fetal liver fibroblast feeder layers and primary mouse embryonic fibroblast feeder cells, found that both the species of feeder layer cells and embryonic treatments were significantly affected the attach rate and growth of GES, Half of the goat in vivo developed blastocysts on the MEF feeder posted the highest attach rate 55.6%, can be passaged 6 times in vitro, and on goat fetal fibroblast feeder layer goat ICM get the lowest adherence rate of 10%, can be passaged 2 times in vitro.2.In order to compare the effect of gelatin and ECM to goat ES cell culture .Plant the half of the in vitro developed embryos on the PMEF feeder layer, with culture medium 1 and found that the adherence rate of ICM in gelatin was 35.7%, the adherence rate of ICM in ECM-coated culture dishes was 40%, can be passage 5 times, the difference was not significant.3.This study compared adhesion rate of the in vitro developed blastocyst and clone blastocyst and subculture, result showed that half embryos in the ECM-coated culture dishes, PMEF feeder layer culture medium 1 adherent rate of clone blastocyst was 25%, can be passage 2 times, in vitro development of blastocyst ICM adherence rate was 40%, can be passage 5 times, the difference was Significant.4.Isolation theprimordial germ cells (PGCs) and the surrounding tissue from Inner Mongolia cashmere goat fetus,cultured in three culture systems, results showed that: The A culture system without any factors can only be passaged one generation, B culture system add only LIF and Insulin could be passage four generations, C culture system adding LIF, Insulin and high-quality fetal bovine serum culture system can be passage 9 generations,5. Identified stem like cells and PGCs by RT-PCR, AKP staining, and immunofluorescence.AKP identified was positive, RT-PCR detection ofβ-actin, hTERT, GAPDH, Nanog, Oct3/4, immunofluorescence detection of Oct3/4, SSEA-3, SSEA-4 were positive. In vitro differentiation culture can found nerve cells, fat cells and epithelial cells like cells更多还原