【作者】 王争光；

【导师】 许梓荣； 俞颂东；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2007， 博士

【摘要】 奶牛体细胞核移植技术在加速品种改良、结合转基因技术生产珍贵医用蛋白等方面有着广阔的应用前景。但目前克隆效率低、流产率高、后代存活率低和出生后代发育异常等成为该项技术广泛应用的瓶颈。导致这一瓶颈的因素很多,其中现有卵母细胞体外培养系统不能使卵母细胞,特别是卵胞质达到成熟和激活方法不能完全激活核移植后的重构胚是两个重要因素,这两个因素能引起核移植后核质互作出现异常,导致重构胚不能正确的实现重编程。因此,完善奶牛卵母细胞体外成熟培养系统,优化核移植后卵母细胞的激活方法,成为提高核移植效率的两个关键环节。本研究依据现代细胞营养学理论,形成了一种无血清、化学成分明确的奶牛卵母细胞体外成熟培养液,并优化该培养液成熟培养的奶牛卵母细胞的激活条件。以体外培养的奶牛耳部成纤维细胞、输卵管上皮细胞及卵丘细胞为供体细胞,进行体细胞核移植证实了上述奶牛卵母细胞成熟培养液和激活方法的可行性。同时,研究绿茶多酚对IVF及NT植前胚胎体外发育的影响及机理,为提高核移植胚胎体外发育率提供理论依据。1.奶牛核移植供体细胞系的建立采用组织块贴壁法从奶牛耳部组织分离成纤维细胞,采用机械法分离奶牛输卵管上皮细胞,利用含血清的DMEM/F12培养液对两种细胞进行原代和续代培养；用胰蛋白酶消化液从卵泡中抽取的卵丘-卵母细胞复合物分离卵丘细胞,利用改良的McCoy’s5a培养液进行原代和续代培养。传代纯化后分析三种细胞的形态特征、体外生长曲线和染色体核型,结果表明,三种细胞的形态正常、体外生长曲线符合正常细胞的一般规律、染色体核型正常,均可作为体细胞核移植的供体细胞。2.无血清、化学成分明确的奶牛卵母细胞体外成熟培养液的优化用PVP-40代替血清,建立了一种化学成分明确的卵母细胞体外成熟基础培养液(SOFaa-PVP-40),分别单独将不同浓度的葡萄糖(0 mM、1.5 mM、5.6 mM和20 mM)、IGF-I(0ng／mL、50 ng/mL、100ng/mL和150ng／mL)、GTPs(0μM、10μM、15μM和20μM)添加到基础培养液中,对奶牛卵母细胞进行IVM、IVF和IVC,以筛选三种物质适宜的添加浓度。结果表明,获得最高卵裂率和囊胚率的葡萄糖、IGF-I和GTPs添加浓度分别为5.6mM,100ng／mL、15μM。将三种物质以优化的浓度同时加入到基础培养液中,构建一种无血清、化学成分明确的优化培养液(Optimized SOFaa),以传统的TCM-199+ FBS培养液为对照,比较两组培养液培养后的卵母细胞体外成熟率、卵裂率、囊胚率及细胞内GSH含量。结果表明,所构建的卵母细胞优化培养液可以代替传统的TCM-199+FBS培养液用于奶牛卵母细胞的体外成熟培养,并提高了成熟后卵母细胞的囊胚发育率(39.2%vs.30.4%,P<0.05)。卵母细胞发育能力的提高与细胞内GSH含量的增加有关(14.3 pmol/oocyte vs.8.9 pmol/oocyte, P<0.05)。本研究的结果还证实,在秋季利用切剖法对卵巢表面直径为3-6 mm的卵泡采集卵母细胞,可以获得较好的卵母细胞数量、体外成熟率、体外受精后卵裂率及囊胚率。3.奶牛卵母细胞激活条件的优化通过比较不同处理对卵母细胞激活及孤雌胚发育的影响筛选奶牛卵母细胞的激活方法。奶牛卵母细胞在Optimized SOFaa培养液中成熟并脱去卵丘细胞后按照试验设计进行激活处理。以单一激活,成熟的卵母细胞用离子霉素(5μM,5min),乙醇(7%,7 min),钙离子载体A23187(5μM,5 min)或锶(10 mM,5h)单独激活试剂进行处理。离子霉素和乙醇处理组获得的原核形成率(39.4%,41.2%vs.21.3%,25.2%)和卵裂率(30.3%,28.1%vs.19.7%,23.7%)显著高与另外两组(P<0.05)。以联合激活,用上述浓度的离子霉素和乙醇与6-二甲基氨基嘌呤(2.0 mM,3h)或放线菌酮(10μg／mL)+细胞松弛素B(7.5μg／mL,3h)联合处理卵母细胞。离子霉素+6-二甲基氨基嘌呤处理组和乙醇+放线菌酮+细胞松弛素B处理组卵母细胞获得的原核形成率(51.0%,53.4%vs.39.6%,35.3%)、卵裂率(41.3%,41.6%vs.28.1%,26.1%)及囊胚率(18.2%,19.3%vs.10.1%,11.1%)显著高于与其他组(P<0.05)。以IVF胚胎为对照,对离子霉素+6-二甲基氨基嘌呤和乙醇+放线菌酮+细胞松弛素B激活获得的孤雌胚胎进行染色体倍性分析和凋亡检测。结果表明,与离子霉素+6-二甲基氨基嘌呤处理组相比,乙醇+放线菌酮+细胞松弛素B处理组获得了较低的单倍体率(7.4%vs.15.5%,P<0.05)和细胞凋亡率(7.0%vs.9.1%,P<0.05)。结果揭示,乙醇+放线菌酮+细胞松弛素B联合激活是更有效的奶牛卵母细胞激活方法。4.优化的卵母细胞体外成熟培养液和激活方法在奶牛克隆技术中的应用为了验证筛选的Optimized SOFaa卵母细胞体外成熟培养液和激活方法用于奶牛体细胞核移植的可行性,将奶牛卵母细胞分别培养于Optimized SOFaa培养液和传统培养液(TCM-199+FBS)中,成熟后分别以卵丘细胞为供核细胞进行核移植,比较两组重构胚的卵裂率及囊胚率,结果表明,Optimized SOFaa可用于核移植受体卵母细胞的成熟培养,并且用该培养液成熟的卵母细胞获得了比传统培养液成熟的卵母细胞更高的囊胚率(21.2%vs.14.3%,P<0.05)。优化的激活策略也可以用于奶牛体细胞核移植。以Optimized SOFaa培养液成熟的卵母细胞为受体,分别以成牛耳部成纤维细胞、输卵管上皮细胞及卵丘细胞为供体,进行核移植,结果表明,供体细胞类型能影响核移植的效率,以卵丘细胞为供体获得的囊胚率显著高于另外两种核供体(21.2% vs.15.9%,13.7%,P<0.05),卵丘细胞更适合于作为奶牛体细胞核移植的供体细胞。分别用GO期卵丘细胞和正常生长的卵丘细胞为供体细胞进行了核移植,结果表明,核质互作能影响核移植的效率,GO期卵丘细胞为核供体获得了较高的囊胚率(21.2% vs.12.2%,P<0.05)。5.绿茶多酚对奶牛IVF和NT胚胎体外发育能力的影响及机理研究IVF和NT胚胎外生产时由于必须在可见光和高于体内的氧气条件下操作,增加了胚胎细胞内活性氧(ROS)的产生。ROS对牛胚胎的毒害作用已经被广泛证实,绿茶多酚(GTPs)是绿茶叶中分离提纯的酚类化合物复合体,研究已经表明GTPs具有强的抗氧化活性,是有效的ROS(如O2-、H2O、OH和NO)清除剂。为了研究GTPs对奶牛卵母细胞体外受精和植前胚胎早期发育的影响。分别在卵母细胞体外受精期间和IVF及NT胚胎培养期间向培养液中加入不同浓度的(0,10,15,20,25μM) GTPs,结果表明,在IVF培养液中添加不同浓度的GTPs均不能提高卵裂率及囊胚率；与对照组相比,添加15μM GTPs到IVF胚胎培养液中显著提高了囊胚率(33.6%vs.24.8%,P<0.05),但更高浓度GTPs(20或25μM)的添加并没有进一步增加囊胚率(21.1%,19.0 vs.24.8%)。同样在NT胚胎体外培养液中添加15μM GTPs也提高了囊胚率(25.1% vs.19.0%)。本研究还进一步证实,培养在含15μM GTPs中的IVF胚胎和NT胚胎早期发育能力的增强与其细胞内GSH含量增加有关。更多还原

【Abstract】 Cow cloning by somatic cell nuclear transfer (SCNT) has the potential to improve the productivity of enormously valuable livestock. It also has the potential for producing transgenic animals that synthesize large quantities of human proteins for use in medicine. However, low efficiency and high abortion rate and early developmental abnormity of offspring were bottlenecks of cloning application. The success of SCNT techniques depends mainly on two factors including oocyte maturation and its activation. Incomplete oocyte maturation, especially cytoplasmic maturation, and incomplete oocyte activation may result in imprecise reprogramming. Therefore, improvement in media for oocyte maturation and optimization of activation protocol become two pivotal processes for considerable improvement in cloning efficiency.Therefore, a serum-free chemically defined maturation media and activation protocols of cow oocytes were optimized in the present study. The SCNT were performed to confirm the feasibility of optimized maturation media and activation protocol taking cow ear fibroblast, oviduct epithelial cells and cumulus cells as nuclear donors. Effects of GTPs on preimplantation development of IVF and NT embryos and its mechanism were also investigated.1. Culture of donor cells for cow cloningThe fibroblasts were separated from cow ear tissue and were cultured in DMEM/F12 media with serum. Oviduct epithelial cells were separated by mechanical method and cultured in the same media as fibroblast. Cumulus cells were separated from cumulus-oocyte complexes (COCs) by trypsin digestion and cultured in the optimized serum-free McCoy’s5a media. Morphological characteristics, growth curves and karyotypes were analyzed after passage and purification of three types of cells. The results showed that the morphological characteristics, growth curves and karyotypes of three types of cells were normal and accorded with growth rules of common cells. Three types of cells could be used as donor in the cloning technique.2. Optimization of serum-free media for cow oocytes maturationA chemical defined basic medium have been constituted by adding PVP-40 instead of serum. The different concentrations of glucose (0 mM、1.5mM、5.6 mM and 20 mM), IGF-I (0 ng/mL、50 ng/mL、100 ng/mL and 150 ng/mL) and GTPs (0μM、10μM、15μM and 20μM) added into the basic medium, respectively. The oocytes were cultured in these medium with glucose, IGF-I and GTPs, respectively. The results showed that the concentration of glucose, IGF-I and GTPs with significantly higher cleavage and blastocyst rates were 5.6 mM,100 ng/mL and 15μM, respectively. Then three substances with the above concentration were added together into basic medium and constituted an optimized medium (Optimized SOFaa), which was serum-free and chemical defined. Oocytes were cultured in control TCM-199+FBS media and optimized media, respectively. The results showed that optimized media cans instead TCM-199+FBS media as cow oocytes maturation. The rate of development to blastocyst of oocytes maturation with optimized media was significant higher than those maturation with control TCM-199+FBS media (39.2% vs.30.4%, P＜0.05). This improvement in development capacity of preimplantation embryos contributes to the increase of intracytoplasmic glutathione concentration (14.3 pmol/oocyte vs.8.9 pmol/oocyte, P＜0.05). The present study also approved that oocytes collected by puncture method from 3-6mm follicles in autumn had higher the number of oocyte per ovary, rate of maturation, cleavage and development to blastocyst.3. Optimization of activation methods of cow oocytesThe activation methods of cow oocytes were optimized through comparing the efficiency of different treatments on the activation and subsequent development of oocytes. Cumulus-free oocytes which matured with optimized SOFaa were activated according to the experiment designs. For activation treatment alone, the matured oocytes were treated with single activation agents including ionomycin (5μM for 5 min), ethanol (7% for 7 min), calcium ionophore A23187 (5μM for 5 min), or strontium (10 mM for 5 h). The pronuclear formation (39.4%,41.2% vs.21.3%,25.2%) and cleavage rate (30.3%,28.1% vs.19.7%,23.7%) were higher significantly in ionomycin and ethanol treatment alone compared to other treatments (P< 0.05). For the combined activation treatment, the same concentrations of ionomycin and ethanol as above were used in combination with 6-dimethylaminopurine (6-DMAP,2.0 mM for 3 h) or cycloheximide (CHX)+cytochalasin B (CB, 10μg/mL for 3 h). The pronuclear formation (51.0%,53.4%vs.39.6%,35.3%), cleavage rate (41.3%,41.6%vs.28.1%, 26.1%) and blastocyst rate (18.2%,19.3%vs.10.1%,11.1%) were higher significantly (P< 0.05) in ionomycin+6-DMAP treatment and ethanol+CHX+CB treatment compared to other treatments. The parthenogenetic blastocysts produced by activation with ionomycin+6-DAMP and ethanol+CHX+CB and in vitro fertilized blastocysts (control group) were examined for apoptosis and ploidy. The ethanol+CHX+CB treatment shown lower significantly blastocyst apoptosis index (7.0%vs.9.1%, P< 0.05) and percentage of haploid (7.0%vs.9.1%, P< 0.05) compared to ionomycin+ 6-DAMP treatment. These results suggested that ethanol+CHX+CB treatment was more favorable method for parthenogenesis of bovine oocytes.4. Test of optimized maturation medium and activation method of cow oocytes in cloningTo examine the feasibility of the optimized SOFaa media and activation method in cow cloning, oocytes were matured with the optimized SOFaa media and control TCM-199+FBS media, respectively and then SCNT were performed taking cumulus cells as donors. The results showed that the optimized media may instead TCM-199+FBS media for maturation of recipient oocytes. The rates of NT embryos development to blastocysts were significant higher in oocytes maturation with optimized media than that maturation with control TCM-199+FBS media (21.2%vs. 14.3%, P< 0.05). The optimized activation protocol was also practicable in cow cloning. To test the effects of types of donors on preimplantation development of NT embryos, SCNT were performed taking ear fibroblasts, oviduct epithelial cells and cumulus cells as donors, respectively. The rates of NT embryos development to blastocysts were significant higher when cumulus cells as donors than when fibroblasts and oviduct epithelial cells as donors (21.2% vs.15.9%,13.7%, P<0.05). The cumulus cells were more favorable type for cow cloning. To examine the effects of cell cycle of donors on efficiency of cloning, the SCNT were done by using GO-stage and non-GO stage cumulus cells as donors respectively. The result indicated that when used GO-stage cells as donors, blastocyst rate was raised significantly (21.2% vs.12.2%, P<0.05).5. Effects of GTPs on development of IVF and NT embryos and approach its mechanismOocytes and embryos are inevitably more exposed to oxygen in vitro, which results in increased production of ROS than in vivo because of the necessary manipulations with transient exposure to atmospheric oxygen and visible light during embryos production by IVF and NT. In bovine, the detrimental effects of ROS to embryo development have been confirmed. Therefore, the addition of antioxidants to IVC media may be pre-requisite for improvement of development competence of embryos by in vitro production. The effects of GTPs on preimplantation development of IVF and NT embryos were investigated. The matured oocytes were fertilized and embryos were cultured in a defined conditioned media with or without GTPs supplementation (0,10,15,20,25μM). The results demonstrated supplementation with GTPs on different concentrations had no improvements in rate of cleavage and blastocyst formation (P＜0.05). Supplementation of the culture media of IVF embryos with 15μM GTPs improved rate of blastocyst formation (33.6% vs.24.8%, P< 0.05), while higher GTPs concentrations (25μM) significantly reduced embryos development (P<0.05). Effects of GTPs on NT embryos were the same as that on IVF embryos. This improvement in development capacity of preimplantation IVF and NT embryos contributes to the increase of intracytoplasmic glutathione concentration.更多还原