【作者】 安立龙；

【导师】 邱怀； 窦忠英；

【作者基本信息】 西北农业大学 ， 动物遗传育种与繁殖， 1999， 博士

【摘要】 胚胎干细胞（Embryonic stem cell,ES细胞）是从早期胚胎内细胞团（inner cell mass.ICM）或原始生殖细胞（Primordial germ cells.PGCs）分离和克隆的具有全能性的细胞。ES细胞在体外分化抑制培养时,可以增殖冷冻,操作和筛选,又可通过胚胎嵌合和细胞核移植参与各种组织（包括生殖腺在内）的发育,形成克隆动物。ES细胞作为一种新型的实验材料,广泛应用于动物克隆、转基因动物的生产、真核细胞基因的表达与调控、人类遗传病动物模型的创建,人类器官移植材料的生产以及细胞分化机制的研究等领域。ES细胞分离与克隆技术与遗传工程和胚胎工程相结合,对于阐明动物生长发育的基本规律、抢救和保护濒危动物遗传资源、建立先进的动物育种技术体系具有重大的理论意义和实践价值。 尽管家畜ES细胞具有广阔的应用前景。但有关技术尚未成熟。本研究以牛和小鼠胚胎为材料,从成纤维细胞的分离与克隆,成纤维细胞的冷冻保存,成纤维细胞饲养层的制备,体外分外抑制培养牛胚胎生长行为,牛ES细胞分离与克隆、牛ES细胞的鉴定以及影响牛ES细胞分离与克隆的因素等方面,对牛ES细胞分离与克隆技术进行系统研究,主要内容包括: 1 犊牛睾丸和小鼠胎儿成纤维细胞分离与克隆技术体系的建立以小鼠胎儿和牛睾丸为材料,以DMEM（低糖）+15%NBS+0.1mMβ—巯基乙醇+0.1μM Na₂SeO₃为培养液,以0.25%胰蛋白酶+0.04%EDTA为消化液,建立了小鼠胎儿成纤维细胞和牛更多还原

【Abstract】 Embryonic stem （ES） cells are pluripotential stem cells derived from animal embryos or primordial germ cells （PGCs） . ES cells can be cultured in vitro and kept undifferentiated. After being reintroduced into embryos, ES cells will involve in the development of tissure and organs of recipient embryos and can be transmitted through the germ line. So,foreign genes can be introduced into animal genome with ES cells, transgenic animal and clone animal can be produced by ES cells. Moreover,the most important application of ES cells is to produce recessive mutation, to understand gene functions by targeted mutagensis, to cre-at animal modle of human genetic disease and to understand the law of mammalian animal development. The object of this research were to bulid the technical system on isolation and clone of bovine ES cells. The main contents were as follows :1. Building the technical system on isolation and clone of murine embryonic and bovine testicular fibroblasts. The conclusions are as follow: ① 15 passage murine embryonic fribroblasts （MEF） and 5 passage new bovine testicular fibroblasts （NBTF） were isolated and cloned in media (1.0g/L glucose DMEM （Gibco） supplemented with 15% NBS, 0. 1mM β-mercaptoethanol（Sigma）, 0. 1μM Na₂SeO₃. The murine embryonic and new bovine testicular tissures were cut and incubated at 37℃ in 0. 25% trypsin / 0. 04% EDTA; ② Compared with MEF, the length and width of NBTF are big, inddition to, NBTF cell grow fast; ③ Murine embryos from 12d to 16d were most suitable for isolationand clone of MEF; （4） Treated with 0. 25%trypsin + 0. 04% EDTA, single MEF cell and single NBTF cell were isolated and cloned by short term （from 2min to 3min）j （5） To supplement with 0. l\iM Na2SeO3, 15% NBS and 0. 1 mM (3-mercaptoethanol is beneficial to reproduction of MEF and NBTF cells.2. Studies on cryopreservation of NBTF and MEFMethords for cryopreservation of MEF and NBTF were investigated in this paper. The results showed as follow: ? The vitality of MEF cells and NBTF cells exposed to the freezing media n （PBS supplemented 0. 4% BSA, 0. 1M sucrose* 1. 5M ethylene glycol（EG）） higher than freezing media I （DMEM + 10% NBS + 10%DMSO）. （2） When NBTF and MEF were freezed in media I , the balanced time can be reduced from 2. 5h to 0. 5h without influence on their vitality. Moreover, freezing rate can be increased from 0. 6°C/min to 1. 2°C/ min, induced ice did not influence on their vitality. On the vitality of NBTF cells and MEF cells, thawing media I PBS （supplemented 0. 1M sucrose, 0. 4% BSA）>thawing media I （DMEM） （supplemented 10% NBS）>thawing media M PBSA .3. Growth behavior of bovine embryos ICM on MEF feeder layerThe growth behavior difference between bovine embryos and murine embryos were studied on MEF feed layer. The results showed as follows: （T） After having spread, bovine embryonic trophoblast formed a transparent and membranous structure coving inner cell mass （ICM）, however, murine embryonic trophoblast formed disc structure} （2） Bovine ICM formed four kinds of colonies with different morphology, including the mass-like, the net-like, the stream-like and the mixtured colonies; ? Compared with murine ICM, the bovine ICM grew more fast, so, at approximately day 3 to day 6 after plating for bovine embryos and day 3 to day 4 after plating for murine embryos, growing ICM were passaged at first j （4） The mixed colonies differented very early, while the others differentiated very late. Most like ICM grew in a defined spot, but stream-like and net like ICM proliferated fast and spread quickly. The single blastomeres from bovine late morula and murine late morula formed subblastophere. When the trophoblast were removed,ICM cell would differentiation rapidly.4. Isolation and clone of bovine ES cellsA total of 30 bovine morula were cultured in tissure medium (DMEM supplemented with 15% NBS, 0. lfiM Na2SeO3, 0. lmM 3 mercaptoethanol（Sigma） on a feeder layer of MEF or NBTF 5 days or 6 days later. The bovine ICM cells were disaggregated by a short-term trypsin treatment. The results sowed as follows: （T） Disaggregation of the ICM : ICM-derived cell embryos were selected following a 5-day to 6-day culture period and disaggregated with trypsin, then the smaller cellular fregment were transferred into a fresh feed layer, formation of 4 type of colonies, including ES-like cell colonies, fibroblast-like cell colonies, epithelioid like cell colonies and trophoblast like cell colonies;（2） Most of colonies derived mass ICM were ES-cells line consisted. However, colonies derived stream ICM were consisted of ES like cells and epitheliod like cells, most of colonies derived net ICM were composed of epithelioid like cells;（3） Compared with mouse ES cells, bovine ES cell grow slowly, so, ES cell were passaged at intervals of 4-days to 5-days. In addition to, bovine ES cell darker than murine ES cell. （4） Supplemented with 15% supper NBS （the ration of mouse embryo hatched are more than 86% within 24h culture）; （5） Compared with the ration of ES colonies among TCM 199, DMEM （high glucose） and DMEM （low glucose）, the ration of embryo hatched of DMEM （L） and DMEM （H） more than TCM 199. as result, The ration of ES colonies cultured in DMEM （L） are more than those cultured in DMEM （H） and in TCM199; （6） Super bovine embryos formed mass ICM, the ration of ES colonies are higher than stream ICM and net ICM derived normal embryos; （7） The ration of clone of ES cell were most high in DMEM （low glucose） culture medium supplemented with 15% NBS, 0. l|iM Na2Se03, 0.1 mM 3-mercaptoethanol, lOOOlU/ml LER and lOng/ml IGF; （8） ICM or ES colonies treated with 0. 05% trypsin and 0. 01% EDTA long term（6min）, the ration clone of bovine ES cell were most high （75%）; （9） 6 passage bovine ES like cell and 9 passage ES like cell were obtained in this research.更多还原