【作者】 安立龙； 窦忠英； 杨奇； 冯秀亮；

【Author】 AN Li-long, DOU Zhong-ying, YANG Qi, FENG Xiu-liang(1 Agriculture college of Zhanjiang Ocean University,Zhanjiang , Guangdong, China 524008, 2 Key laboratory ofAnimal Reproductive Endocrinology and Embryo Biotechnology of Agricultural Ministry of China’,NorthwesternScience &Technology University of Agriculture & Forestry, Yangling, Shaanxi, China, 712100)

【机构】 湛江海洋大学动物科学系； 西北农林科技大学国家干细胞研究中心陕西省分中心；

【摘要】 胚胎干细胞是来自于早期胚胎内细胞团或胎儿原始生殖细胞的一种具有潜在发育全能性的细胞,这种细胞在分化抑制条件下可以无限增殖,去分化抑制后可以诱导形成各种组织细胞。在分化抑制条件下早用胚胎发育状况对源于内细胞团的胚胎干细胞的分离与克隆效率具有重要的影响。本文研究了分化抑制物对体外培养的牛和小鼠胚胎生长发育的影响,分析了白血病抑制因子和胰岛素生长因子以及葡萄糖含量对对体外培养的牛和小鼠胚胎生长发育的影响,结果表明:将牛和小鼠胚胎培养于含有15%NBS, 0.1mmol/lβ-二巯基乙醇,2mmol/l谷氨酰胺,1mmol/l丙酮酸钠,0.1mmol/l必需氨基酸和0.1mmol/l Na₂SeO₃的低糖DMEM（葡萄糖1.0g/L）溶液的小鼠胎儿成纤维细胞饲养层中,牛囊胚在体外培养后36-60小时开始孵化（脱透明带）,在体外培养后96小时开始贴附小鼠胎儿成纤维细胞饲养层,牛桑椹胚脱透明带和开始贴附小鼠胎儿成纤维细胞饲养层时间比囊胚迟12-24小时。小鼠囊胚在体外培养后24-48小时开始孵化（脱透明带）,在体外培养后36-40小时开始贴附小鼠胎儿成纤维细胞饲养层,小鼠桑椹胚脱透明带和开始贴附小鼠胎儿成纤维细胞饲养层时间比囊胚迟12-24小时。体外分化抑制培养的牛胚胎滋养层细胞与内细胞团连接不紧密。迅速形成囊状结构,覆盖于内细胞团上;体外分化抑制培养的小鼠胚胎滋养层细胞与内细胞团连接紧密,形成盘状结构。源于牛胚胎的内细胞团在小鼠胎儿成纤维细胞饲养层培养,出现了团状、条状、网状和混合型等形状,实验证明,分离和克隆牛类ES细胞效率由高到低的次序依次是团状、条状、网状和混合型ICM,细胞分化程度由高到低次序依次也是团状、条状、网状和造合型ICM。与小鼠胚胎内细胞团相比较,体外分化抑制培养的牛胚胎内细胞团发育比较迟缓,体外分化抑制培养牛囊胚ICM初次传代时间为培养后5-6天,小鼠囊胚ICM初次传代时间为培养后3-4天。牛和小鼠胚胎在体外分化抑制条件下培养,胚胎附着率与ICM形成率由高到低依次是卵化胚、囊胚和桑椹胚。与全胚相比较,牛和小鼠裸胚贴壁时间提前,但ICM形成率和ICM的发育与分化状况均无差异。牛和小鼠桑椹胚卵裂球（中、晚期）在分化抑制条件下培养,形成亚囊胚而不能形成ES细胞。剥离牛滋养层的内细胞团在体外分化抑制条件下培养,有分化增加的趋势。在体外分化抑制培养中,ICM增殖速度由大到小依次是高精DMEM（葡萄糖4.50g/L）,低糖DMEM（葡萄糖1.0g/L）和TCM199溶液。10ug/L IGF和1000IU/mLLIF可以促进小鼠与牛胚胎ICM生长,抑制细胞分化。更多还原

【Abstract】 Embryonic stem cells is derived from inner cell mass (ICM) of the early embryos or primordial germ cell (PGCs) , which they not only keep pulripotency of development but also proliferate unlimited under the conditions of inhibition differentiation in vitro. The development of embryos for animals play an important role in isolating and cloning ES derived from inner cell mass. The state of development of bovine embryos and murine embryos in culture system including inhibitor of cell differentiation were observed. Furthermore, We have studied the effect of cytokine such as LIF and IGF on growth behavior between Bovine embryos and Murine embryos in vitro. The bovine and Murine embryos including morula ,blastula , hatched blastula and blastomere were cultured respectively on PMEF feeder with DMEM supplement with 15%NBS, 0.1 mmol/1 P -mercaptoethanol , 2m mol/1 glutamine,lm mol/1 sodium pyruvate, 0.1 m mol/1 nonessential amino acids and 0.1 m u mol/1 Na2SeO3 . The results showed as follows: the blastocyst of bovine begain to hatche after culturing for 36-60 h, and attached on the murine embryos fibroblast cell (MEF) feeder layer after culturing for 96-120 h. The time that the morulae of bovine began to hatche and attached on the MEF feeder layer were later 12-24 h than those of the blastocyst of bovine, the blastocyst of murine begain to hatche after culturing for 12-24 h, and attached on the murine embryos fibroblast cell(MEF) feeder layer after culturing for 24-48 h. The time that the morulae of murine began to hatche and attached on the MEF feeder layer were later 12-24 h than those of the blastocyst of murine. With embryos having attached, Bovine embryonic trophoblast formed a transparent membranous structure covering on inner cell mass (ICM), however, Murine embryonic trophoblast formed disc structure; Bovine embryos formed four kinds of ICM colonies with different morphology including the mass like, the net like, the stream like and the mixture like colonies; Compared with Murine ICM, the bovine ICM grew more slow, so, the Bovine ICM was passaged at first after culture of approximately 5 - 6 d in vitro , and but murine ICM was passaged at first after attachment of 3 - 4 d on PMEF feeder layer; The mixture colonies of Bovine ICM differentiated very early, while the others differentiated very late. Most ICM like mass of Bovine grew in a defined spot, but bovine ICMs like stream and ICMs like net proliferated fast and dispersed quickly. We found that the single blastomeres derived from late bovine morula and late murine morula formed subblastophere, moreover,Bovine ICM cell would be differentiation rapidly if the trophoblast were removed. The medium that growth speed of ICM for Bovine and Murine from large to small developed were high glucose DMEM (4.5 g/L glucose), low glucose DMEM (1.0 g/L glucose) and TCM199, The medium containing 1000 lU/mL LIF and 10 ng/mL IGF were benefit to development of ICM and ES cells for Bovine and Murine.更多还原