【作者】 王凌燕；

【导师】 李德雪；

【作者基本信息】 吉林大学 ， 基础兽医学， 2009， 博士

【摘要】 体细胞核移植动物的成功是几十年来生物学领域的重大突破之一。核移植技术为研究配子和胚胎发生、细胞和组织分化、基因表达调控、核质互作等机理提供了平台。金黄地鼠是一种较好的人类疾病动物模型,对金黄地鼠进行体细胞核移植具有重要的意义。本人对金黄地鼠体细胞核移植及其相关技术问题进行了研究,其中包括金黄地鼠卵母细胞的获得、成熟卵母细胞的活化、卵母细胞的老化,以及显微操作法去核过程的简化、供核体细胞细胞系的建立、供核细胞与卵母细胞的融合及克隆胚胎构建的方法。结果发现:(1)电刺激和6-DMAP均可促使金黄地鼠卵母细胞卵裂,低强度电脉冲和6-DMAP联合作用能使绝大多数的卵母细胞发生卵裂;(2)体内成熟和体外培养的金黄地鼠卵母细胞呈现出明显的老化状态,而TSA和咖啡因能够有效的延缓其衰老;(3)第一极体的状态及其与卵母细胞染色体的位置关系随着卵龄和培养条件的变化而变化;(4)秋水仙素和秋水酰胺诱导的金黄地鼠卵母细胞化学去核和化学辅助去核效果显著,明显高于盲吸去核率;(5)金黄地鼠卵母细胞与胎儿成纤维细胞和卵丘细胞可以在电刺激条件下发生融合。总之,本论文对金黄地鼠体细胞核移植中的重要技术环节进行了有益探讨和论述,为获得克隆地鼠奠定了坚实基础。更多还原

【Abstract】 The success of cloning animals is the great improvement for biological research. The technology of nuclear transfer is very useful in many researches, such as the gametogenesis, cell differentiation and genetic expression and regulation, et al. Cloned offspring have been produced by somatic cell nuclear transfer (SCNT) in many mammalian species. The golden hamster is an excellent model animal for many research fields. It represents an attractive species for studies ranging from reproductive physiology, oncology, genetics and virology. In order to establish animal models for human diseases using golden hamsters by somatic cell neclear transfer, this study was conducted.The activation conditions of golden hamster oocytes were examined and optimized. Golden hamster oocytes of different ages were tested with three treatments: (1) an electrical pulse ranging from 10V/mm to 600V/mm; (2) incubation for 2h to 6h in 2mM of 6-DMAP; (3) combination of electrical and 6-DMAP stimulus. Our results demonstrated that oocytes exhibited an intensity-dependent increase in cleavage from 10V/mm to 500V/mm, but younger oocytes (13.5h post hCG) could not bear pulse higher than 500V/mm; the highest cleavage rates (46%) of 15h-post-hCG oocytes were observed with 4h 6-DMAP treatment, but 6-DMAP had difficulty to induce cleavage of 17h and 19h post-hCG oocytes. Under combined treatment, the cleavage of oocytes rates reached to 96.49%.Ageing and anti-ageing of golden hamster oocytes were studied. Oocytes were collected under three different conditions: (1) oocyte direct recovery from the oviduct of hormonally treated donor at different age; (2) oocyte recovery from the oviduct of hormonally treated donor (15h post hCG) followed by 5h, 10h, 20h in vitro culture with or without cumulus cells; (3) 15h-post-hCG oocytes treated with TSA and caffeine for 5h to 20h. Then the spontaneous parthenogenetic activation, cortical granules and developmental potential of oocytes were investigated. Our results demonstrated In vivo matured oocytes were ageing with the age, and spontaneous parthenogenetic activation reached to 36.96% in oocytes of 35h post hCG; cumulus cells had little effect on ageing of in vitro-cultured oocytes; TSA and caffeine could delay the ageing process, the developmental potential of oocytes in different ages was higher than that of untreated oocytes; cortical granules stained were decreased with age.Changes in the reciprocal position of the first polar body (FPB) and chromosome set of MII oocytes of golden hamsters were examined. The oocytes were collected under three different conditions: oocytes of in vivo; oocytes of in vitro culture; oocytes of in vitro maturation; oocytes treated with colchicine and/or cytochalasin B. Then denuded oocytes were stained with Hoechst 33342 and propidium iodide and evaluated under an inverted fluorescence microscope and a confocal laser scanning microscope. Our results demonstrated that 82.10% FPBs of 13.5h-post-hCG oocytes were in the zone of 0o-30o, the change in FPBs position increased with age in in vivo-matured oocytes; cumulus cells could protect the FPBs of in vitro-cultured oocytes from degeneration, while they did not significantly affect the repositioning of FPBs; In vitro-matured oocytes aged slower, still 77.94% FPBs were in the zone of 0o-30o when they were cultured for 18 h; The oocytes could be induced cytoplasmic protrusions in colchicine, while the cytochalasin B almost had no effect on FPBs repositioning.Concentrations of colchicine and demecolcine for inducing cytoplasmic protrusion (containing chromosomes) and assisting enucleation of golden hamster oocytes were examined. Denuded oocytes of different ages were treated with different concentration of colchicine and demecolcine. Then cytoplasmic protrusions of oocytes were removed with a micromanipulation pipette. Our results demonstrated that: after 13.5-18h post hCG injection, approximately 90% of oocytes treated with 10μg/ml of colchicine for 1h had formed cytoplasmic protrusions, and some 13.5h-post-hCG oocytes could be induced enucleation (23.25%) directly as the chromosomes eliminated with the second polar body; when treated with 0.4μg/ml of demecolcine for 1h, the cytoplasmic protrusion rate of oocytes reached nearly 100%; when the cytoplasmic protrusions induced by colchicine or demecolcine were removed, assisted enucleation rates were over 80%, much more successful than 31.53% of blind enucleation.Fetuses fibroblasts and cumulus cells of golden harmsters were collected as nuclear donor cells in nuclear transfer. The 10-days fetuses were collected and digested to prepare golden harmster fibroblasts. Cumulus cells were collected when the cumulus–oocyte complexes being treated with hyaluronidase. The oocytes were enucleated and electrofused with donor cells. Then the fusion efficiency was examined 30min after treatment. Our results demonstrated that 22.14% fibroblasts and 16.59% cumulus cells were fused with enucleated oocytes.In conclusion, in this study conditions of oocytes activation and cleavage had been optimized; spontaneous parthenogenetic activation, cortical granules, developmental potential and reciprocal position of the FPB were observed in oocytes ageing process; colchicine and demecolcine were used in assisting enucleation of hamster oocytes; hamster cloned embryos were constructed by cell fusion. In one word, all of these experiments were useful to establish animal model for human diseases using golden hamsters.更多还原