【作者】 丁向彬；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2008， 博士

【摘要】 体细胞核移植的成功说明了高度分化的细胞仍具有支持发育的全能性,卵母细胞胞质中存在着将分化体细胞重编程至多能胚胎细胞状态的全部因子。但是,体细胞核移植的成功率却非常低,而且克隆动物存在不同程度的发育缺陷。目前普遍认为,体细胞核移植的低成功率以及发育异常和供体细胞核的不完全表观遗传重编程有关。DNA甲基化和组蛋白乙酰化是哺乳动物主要的表观遗传修饰形式,是调节基因组功能的重要手段,在胚胎的正常发育过程中具有显著的调控作用。本文对牛体外受精(IVF)胚胎和体细胞核移植(SCNT)胚胎附植前各个发育阶段的DNA甲基化和组蛋白乙酰化状态进行分析,找出体细胞克隆胚胎在基因组甲基化和组蛋白乙酰化水平上与IVF胚胎的差异,研究表观遗传状态对克隆胚体外发育能力的影响。1.研究卵母细胞成熟培养液中添加表皮生长因子(EGF)和胰岛素(Insulin)对其体外发育的影响,精子的不同处理方法对卵泡卵母细胞体外受精的影响以及细胞共培养系统对体外受精胚胎体外发育的影响。结果表明,成熟培养液中添加30 ng/mL EGF可以显著提高卵母细胞的成熟率和受精后的卵裂率;精子经上浮法处理后可以得到较高的卵裂率和囊胚发育率;输卵管上皮细胞共培养可以显著提高受精后的囊胚发育率,可以用于牛IVF胚胎的体外培养。2.探讨了不同激活方法对牛核移植胚胎激活效果和体外发育的影响,以及细胞共培养体系对牛体细胞核移植胚胎体外发育的影响。结果表明,5μmol/L离子霉素联合2mmol/L 6-DMAP激活牛核移植胚胎的效果比较好,可以得到较高的卵裂率和囊胚发育率;输卵管上皮细胞饲养层共培养可以显著提高重构胚的囊胚发育率,比较适合作为体细胞核移植胚胎发育的共培养体系。3.采用免疫荧光染色技术,对牛IVF胚胎和SCNT胚胎附植前各个发育阶段的DNA甲基化和组蛋白乙酰化状态进行检测,研究克隆胚胎在基因组甲基化和组蛋白乙酰化水平上与IVF胚胎的差异。结果表明,克隆胚胎各个发育阶段DNA甲基化水平均高于体外受精胚胎,其中2-细胞到桑椹胚阶段甲基化水平显著高于IVF胚胎,从头甲基化时间提前;克隆胚胎4-细胞到囊胚阶段乙酰化水平低于体外受精胚胎,其中8-细胞阶段乙酰化水平显著低于体外受精胚胎。与体外受精胚胎相比,牛核移植胚胎附植前各阶段存在异常的DNA甲基化和组蛋白乙酰化模式。4.用DNA甲基化酶抑制剂(5-aza-dC)处理牛供体细胞和克隆胚胎,探讨DNA甲基化水平变化与克隆胚胎体外发育之间的关系。结果表明,低浓度的5-aza-dC(20nM,72h)处理供体细胞或者重构胚不能显著提高克隆胚胎的体外发育率,对克隆胚的表观遗传状态没有明显影响;而供体细胞和重构胚均用低浓度5-aza-dC(20nM,72h)处理可以显著提高囊胚发育率和囊胚细胞数,2-细胞阶段DNA甲基化水平降低,8-细胞阶段的DNA甲基化水平显著降低且与IVF胚胎相比差异不再显著,但组蛋白乙酰化水平没有明显改变。说明克隆胚胎DNA甲基化水平影响其体外发育,降低2-细胞和8-细胞阶段的DNA甲基化水平可以提高克隆胚胎的体外发育能力。5.用组蛋白去乙酰化酶抑制剂(TSA)处理牛供体细胞和克隆胚胎,探讨组蛋白乙酰化变化与克隆胚胎体外发育的关系。结果表明,供体细胞经50nM TSA处理12h可以显著提高克隆胚胎的囊胚发育率,而且2-细胞阶段的组蛋白乙酰化水平升高;TSA处理重构胚不能显著提高克隆胚胎的体外发育率,对重构胚的表观遗传状态没有显著影响;供体细胞和重构胚均用低浓度TSA(50nM,12h)处理可以显著提高克隆胚胎的囊胚发育率,8-细胞阶段的组蛋白乙酰化水平显著增加且与IVF胚胎相比差异不再显著,但TSA处理对DNA甲基化水平没有明显影响。说明克隆胚胎组蛋白乙酰化水平影响其体外发育,增加2-细胞和8-细胞阶段组蛋白乙酰化水平可以提高克隆胚胎的体外发育能力。6.联合应用5-aza-dC和TSA处理牛供体细胞和重构胚,研究两种试剂联合处理对重构胚发育的影响,以及对重构胚甲基化水平和乙酰化水平的影响。结果表明,5-aza-dC(20nM, 72h)联合TSA(50nM, 12h)处理供体细胞和重构胚可以更明显的提高克隆胚胎的体外发育能力,联合处理不但显著降低了2-细胞和8-细胞阶段DNA甲基化水平,而且显著增加了组蛋白的乙酰化水平,从而使克隆胚附植前各发育阶段的表观遗传状态更接近与体外受精胚胎。说明异常的DNA甲基化和组蛋白乙酰化模式不利于克隆胚的体外发育,消除这种异常可以提高克隆胚的体外发育能力。进一步的实验表明,5-aza-dC和TSA对核移植胚胎体外发育的影响没有供体细胞特异性。更多还原

【Abstract】 Adult somatic cell nuclei can be reprogrammed into embryonic state through nuclear transfer, cytoplasm of oocyte possesses factors essential for converting differentiated somatic nuclei into pluripotent status, however, this reprogramming process is of low efficiency mainly due to incomplete somatic nucleus epigenetic remodeling. The abnormal epigenetic modifications include DNA methylation and histone acetylation that serve as monitor of chromatin structure and gene expression. In this paper, the DNA methylation and the histone acetylation status of IVF and SCNT embryos were analysed and compared, effects of epigenetic reprogramming on the development of cloned embryos were investigated.1. In this study, effects of EGF and Insulin, different sperm treatment methods, and different somatic cell co-culture systems on bovine in vitro fertilization (IVF) were investigated. The results showed that, the matured rate and cleavage rate were significantly increased after added 30 ng/mL EGF to in vitro culture medium; the swim-up method resulted in the best cleavage rate and blastocyst rate; and the blastocyst rate was significantly increased when the fertilized oocytes were cultured with bovine oviduct epidermic cells.2. In this study, effects of different activation methods and somatic cell co-culture systems on in vitro development of cloned bovine embryos were investigated. The results showed that, the cleavage rate and blastocyst rate were promoted after the fused couplets acticated with 5μmol/L ionomycin followed by 2mmol/L 6-DMAP; and the blastocyst rate was significantly increased when the cloned embryos were cultured with bovine oviduct epidermic cells.3. The DNA methylation and histone acetylation status of IVF and SCNT embryos were analysed by immunodetection method, and the differences between two kinds of embryos were investigated. The results showed that, the DNA methylation level of cloned embryos were higher than that of IVF embryos, wich was significant from 2-cell to blastocyst stage, and the precocious de novo methylation was found in cloned embryos; the histone acetylation level of cloned embryos were lower than that of IVF embryos, which became significant in 8-cell stage. The results revealed that the DNA methylation and histone acetylation status were abnormal in cloned embryos.4. The epigenetic modification status of cloned embryos were analysed after donor cells and cloned embryos were treated with 5-aza-dC, and the relationship between DNA methylation and the development of cloned embryos were investigated. The results showed that, the in vitro development potential and epigenetic modification status of cloned embryos were not changed after the donor cells or cloned embryos were treated with low concentration of 5-aza-dC (20nM, 72h); when both donor cells and cloned embryos were treated with 5-aza-dC (20nM, 72h), the blastocyst rate and blastocyst cell number were significantly increased, and the DNA methylation level of 2-cell embryos was reduced, which became significant at 8-cell stage, however, the histone acetylation level of cloned embryos was not changed. The results revealed that the DNA methylation status was important for embryo development , and reduce the DNA methylation level at 2-cell and 8-cell stages could promote the development potential of cloned embryos.5. The epigenetic modification status of cloned embryos were analysed after donor cells and cloned embryos were treated with TSA, and the relationship between histone acetylation and development of cloned embryos were investigated. The results showed that, the blastocyst rate of cloned embryos was significantly increased after donor cells treated with 50nM TSA(12h), and histone acetylation level of 2-cell embryos was increased; the in vitro development potential and epigenetic modification status of cloned embryos were not changed after the cloned embryos were treated with TSA; when both the donor cells and cloned embryos were treated with 50nM TSA(12h), the blastocyst rate was significantly increased, and the histone acetylation level of 8-cell embryos was dramatically increased and more closer to IVF embryos, however, the DNA methylation level of cloned embryos were not changed. The results revealed that hisone acetylation status of cloned embryos was important for embryo development , and increase the hisone acetylation level at 2-cell and 8-cell stages could promote the development potential of cloned embryos.6. In this study, the donor cells and cloned embryos were treated with TSA and 5-aza-dC, the following epigenetic status of cloned embryos were analysed and the effects of these reagents on development of cloned embryos were investigated. The results showed that, 5-aza-dC(20nM, 72h) combined with TSA(50nM, 12h) could significantly promoted the development potential of cloned embryos, the DNA methylation level of 2-cell and 8-cell embryos was significantly reduced while the histone acetylation level significantly increased, which became more closer to IVF embryos. The results revealed that the abnormal epigenetic status of cloned embryos could reduce the subsequent development ability, whereas change the abnormal status could promoted the subsequent development. Further studies demonstrated that the beneficial effects of TSA and 5-aza-dC on cloned embryos development were not donor cell specific.更多还原