【作者】 邸科前；

【导师】 李相运；

【作者基本信息】 河北农业大学 ， 动物遗传育种与繁殖， 2007， 硕士

【摘要】 胚胎干细胞由于其独特的生物学特性，在体外研究胚胎的发生发育、新基因的发现、药物筛选和致畸实验，以及作为组织移植、细胞治疗和基因治疗的细胞源、克隆动物、转基因动物生产、发育生物学、动物和人类疾病模型、组织器官的修复和移植等方面有着诱人的前景。四倍体胚胎补偿技术已经成为一个基本的实验技术，虽然四倍体胚胎只能发育到怀孕中后期，但是四倍体与二倍体嵌合体广泛用于研究胚外发育缺陷的相关基因功能。为了充分利用小鼠胚胎干（ES）细胞，就必须从众多小鼠品系中分离ES细胞系。本实验的研究目的是分离不同的ES细胞系并通过制备畸胎瘤和四倍体胚胎补偿技术来检测ES细胞的多能性。本实验以小鼠胎儿成纤维细胞作为饲养层，利用高糖DMEM作为基础培养基，同时添加15％的新生犊牛血清、0.1mM的2-巯基乙醇、1％的非必需氨基酸、2mM的谷氨酰胺和青、链霉素各50IU／ml、LIF（白血病抑制因子）1000IU／ml。5％CO₂、饱和湿度、37℃培养。本研究从58枚（（C57BL／6J×129／Sv）F1）小鼠囊胚内细胞团分离获得5个ES细胞系，从63枚（C57BL／6J×DBA／2）F1×129／Sv小鼠囊胚内细胞团分离获得6个ES细胞系。把ES细胞注入不同品系的小鼠皮下、睾丸质膜下及。肾囊下，均得到了畸胎瘤，且畸胎瘤包含3个胚层的衍生物。我们发现，睾丸质膜下移植ES细胞，似乎更容易长出畸胎瘤，因此我们认为，睾丸质膜下移植ES细胞可能是检测ES细胞多能性的一个简便易行的方法。采用100V／mm融合电压，2×50μsec脉冲时程，对1979枚2细胞胚胎进行电融合，84.8％的胚胎发生融合。通过移植四倍体胚胎和二倍体胚胎来比较两种胚胎发育潜力。将四倍体胚胎和二倍体胚胎分别随机移植到左右两侧子宫角，得出结论：四倍体胚胎与二倍体胚胎附植率没有显著差异，四倍体胚胎前期的发育能力要优于二倍体。两侧子宫角的胚胎附植率没有区别。四倍体胚胎发育到12-13 d后开始死亡，个别发育到14-15 d。实验没有得到出生的四倍体个体。融合的胚胎培养24 h后，86％的融合胚胎形成4细胞或4细胞以上胚胎。将四倍体4细胞胚胎分别与5个（C57BL／6J×129／Sv）F1 ES细胞系聚合后得到1122枚嵌合囊胚，移植假孕51只ICR母鼠，5只妊娠。从5个ES细胞系中共得到了1只ES小鼠，只存活1天，出生后未能哺乳，腹部有病变，肉眼可见鼓胀。胎儿出生重2.8g。ES细胞的遗传背景和生长状态、嵌合方法、体外培养环境、移植方法等都是影响获得ES小鼠的因素。通过四倍体胚胎聚合技术可以简便而快速地从遗传修饰的杂种ES细胞中获得完全由ES细胞发育而来的个体，为基因功能和人类疾病动物模型的研究提供了一条有效途径。更多还原

【Abstract】 Embryonic stem cells （ESCs）, derived from preimplantation embryos, areundifferentiated, immortal cells capable of differentiating into derivatives of all threeembryonic germ layers. Pluripotential ES cells have been used extensively in studies ofembryogenesis, gene expression and regulation, seek the mechanism of cell differentiation,the cell repair and the transplantation of cells, tissues and organs. The production oftetraploid embryos has become a common experimental manipulation in the mouse.Although development of tetraploid mice has generally not been observed beyondmidgestation, tetraploid:diploid chimeras are widely used as a method for rescuingextraembryonic defects and elucidating gene function in vivo. For utilization of the mouseembryonic stem （ES） cells for various purposes, it is desirable that the cell lines areestablished from various sources such as inbred and outbred mouse strains. The majorobject of this paper is to generate mouse embryonic stem cell lines from（C57BL/6J×129/Sv）F1 and （C57BL/6J×DBA/2）F1×129/Sv mouse and tested their abilityto produce completely ES cell-derived mice at early passage numbers by tetraploid embryoaggregation.Three days after ovulation, 58 blastocysts from （C57BL/6J×129/Sv） F1 mice and 63blastocysts from （C57BL/6J×DBA/2）F1×129/Sv mice were recovered by a surgical uterineflush technique and played on passage 2 to passage 5 mouse embryonic fibroblasts feederlayer mitotically inactivated with mitomycin C （1mg/100ml for 3 hours）. After 4-6 days ofgrowth, the intact inner cell mass （ICM） was separated with 0.25% trypsin-EDTA andagain seeded on feeder in Dulbecco’s modified Eagle medium （4500mg of glucose per liter）supplemented with 15% newborn bovine serum, 0.1mM nonessential amino acids, 0.1mM2-mercaptoethanol, 2mM glutamine, 50 units/ml of penicillin, and 50 units/ml ofstreptomycin,1000IU/ml LIF（Leukemia Inhibitory Factor）. Cultures were grown in 5%CO₂, 95% humidity and 37℃in incubator. After 6 to 7 days of culture, Eleven ES celllines were selected and explanted.ESCs were injected to different position: Subcutaneous implant, under kidney andcapsule testes membrance. We gain derivatives of all three embryonic germ layers. We alsoobserved that implant the ESCs under the testes membrance was a simple, efficientprocedure for testing the pluripotency of ESCs.The oviducts of superovulated （C57BL/6J×DBA/2）F2 black females were flushed 44-46 hours after treatment with human chorionic gonadotropin to collect 1979 latetwo-cell-stage embryos. The embryos were placed twenty at a time between two platinumelectrodes laid 1mm apart in 0.3M mannitol supplemented with 0.1mM CaCl₂ and 0.1mMMgCl₂ in the electrode chamber. The blastomeres were fused by two short electric pulse（100V for 50μsec） applied by a pulse generator. Fusion of blastomeres should becompleted in 10-30 minutes.The developmental ability of duple embryos and the tetraploid embryos were assessedby explanted in the mice oviduct. We conclude that there was no significant difference ofthe developmental ability between the duple embryos and the tetraploid embryos. Thetetraploid embryos could develop 12-13 days, several could develop 14-15days. Ultimately, we did not gain the alive tetraploid foetus.After 24 hours of culture, most of the fused embryos developed to the four-cell stage.Zonae pellucidae of the 1444 four-cell-stage fused embryos were removed by treatmentwith 0.5% Pronase solution. The embryos were aggregated with five ES cell lines. After 24hours of aggregation coculture, 1122 embryos aggregated with ES cells were collected andtransferred into the uteri of 51 pseudopregnant recipients. Five recipients were pregnant.These pregnant recipients were routinely subject to a Caesarean section or spontaneousdelivery and got 1 fetus derived from five ES cell lines, designated B1, B2, B3, B4, B5. Inresult, the only one ES cell-tetraploid neonates derived from B4 ES cell lines survived onlyone day. ES cell-tetraploid pups obtained from hybrid ES cell displayed increased placentaland birth weights.The present results prove that ES cell-tetraploid mice （ES mice） are completelyderived from embryonic stem cells and can be obtained upon aggregation of hybrid EScells and tetraploid embryo. Genetic heterozygosity is a crucial parameter for postnatalsurvival of mice that are entirely derived from ES cells by tetraploid embryocomplementation. This method represents a simple, efficient procedure for immediategeneration of targeted mouse mutants from genetically modified ES cell clones, in contrastto the standard protocol, which involves the production of chimeras and several breedingsteps.更多还原