【作者】 杨月春；

【导师】 方南洙；

【作者基本信息】 延边大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 延边黄牛作为我国五大地方良种牛之一,其肉质细嫩,营养丰富,具有优秀的肉用潜能和广阔的产品商业化发展前景,随着我国畜牧业的发展和吉林省“东黄西红”肉牛发展战略的实施,延边黄牛养殖业发展越来越快,扩大产业化规模和提高科技附加值已经成为当前延边黄牛肉用发展的一个必要途径。体细胞克隆技术作为一种新兴的科技手段,不仅能够作为胚胎干细胞技术、转基因技术和医学等科学研究的技术支撑,同时在畜牧业上可以用来生产大量具有相同基因型的家畜,在家畜育种、保种上具有深远的意义,但是目前为止的研究表明,该项技术存在效率低、生产的后代多有生理缺陷等情况,还不能够达到生产实际的需要。在体外受精、人工授精、胚胎移植等繁殖生物技术中,研究配子在体外生产或体外保存过程中所受的氧化损伤,在体细胞克隆技术中研究显微操作方法、融合激活方法、核质重组(核重编程)机制、DNA甲基化等方面已有大量报道,研究体细胞克隆后代生理缺陷的分子机制已经成为新的热点,但是关于体细胞克隆胚胎的氧化损伤研究尚未见报道。目的：(1)观察氧化剂过氧化氢(H2O2)对延边黄牛体细胞克隆胚胎体外发育的影响,探讨过氧化氢对其氧化损伤的作用；(2)观察抗氧化剂谷胱甘肽(GSH)对延边黄牛体细胞克隆胚胎体外发育的影响,探讨谷胱甘肽对其氧化损伤的保护作用；(3)以孤雌激活胚胎为对照,检测延边黄牛体细胞克隆胚胎内部H2O2和超氧阴离子(O2·-)两种活性氧(ROS)的含量,探讨其在延边黄牛体细胞克隆胚胎体外发育不同阶段的变化规律；(4)以孤雌激活胚胎为对照,检测延边黄牛体细胞克隆胚胎不同发育阶段编码过氧化氢酶(CAT)、含锰的超氧化物酶(Mn-SOD)和谷胱甘肽过氧化物酶(GPx)的mRNA表达水平,探讨其与氧化损伤程度之间的关系。方法与内容：(1)在体外培养液中添加外源性H2O2,观察延边黄牛体细胞克隆胚胎体外发育效率、发育速度以及氧化损伤带来的质量影响,确定H2O2影响其发育的临界浓度、影响最大的时间；(2)在体外培养液中添加外源性GSH,观察延边黄牛体细胞克隆胚胎体外发育效率,确定缓解氧化损伤所需添加的适宜浓度及适宜时间；(3)使用二氢乙锭和2′,7′-二氯荧光素二乙酸酯分别对延边黄牛体细胞克隆胚胎内部的H2O2和O2·-进行染色,使用荧光显微镜观察照相,利用图像处理软件进行量化分析,测定体细胞克隆胚胎不同发育时期的ROS含量,探求体细胞克隆胚胎内部活性氧的变化规律；(4)使用特异引物,利用反转录聚合酶链式反应扩增延边黄牛体细胞克隆胚胎编码CAT、Mn-SOD和GPx的mRNA,并通过琼脂糖凝胶电泳检测其表达结果,同时使用凝胶图像分析软件进行半定量分析,确定其在延边黄牛体细胞克隆胚胎体外不同发育阶段的表达规律。结果：(1)添加0μMol/L、60μMol/L、80μMol/LH2O2时卵裂率显著高于100μMol/L和120μMol/L组(P<0.05),所有添加H202的处理组囊胚发育率显著低于未添加的处理组(P<0.05)；0～24h添加H2O2的处理组卵裂率显著高于其它组(P<0.05),48～72h组16细胞以上发育率显著低于其它处理组(P<0.05)；不同时期不添加H202对延边黄牛重组胚卵裂率没有显著影响(P>0.05),但是不同时期不添加H2O2时所有处理组16细胞以上发育率显著低于对照组(所有时期不添加,P<0.05)；经过H2O2处理后的重组胚卵裂球表面粗糙,胞质不均匀,少且疏松,偶尔能见到极少或没有胞质的卵裂球,卵裂球与透明带之间的间隙较大,发育不规则,卵裂球大小不一,相差较大,存在大量细胞碎片。(2)添加外源性GSH时,1 mMol/L组卵裂率显著高于7 mMol/L组(P<0.05),1 mMol/L和7 mMol/L组卵裂率与0 mMol/L和4 mMol/L组差异不显著(P＞0.05),1 mMol/L组囊胚发育率显著高于0 mMol/L、4 mMol/L和7 mMol/L组(P<0.05)；不同时间添加GSH时,0～24 h组和24～48 h组卵裂率显著高于对照组、48～72 h组和72～96h组(P<0.05),16细胞以上发育率与其它组差异不显著(P>0.05),对照组16细胞以上发育率显著低于48～72 h组和72～96h组(P<0.05)。(3)2细胞期孤雌激活与体细胞克隆组间O2·-水平差异不显著(P>0.05),4细胞期、8细胞期、16细胞期和囊胚期体细胞克隆组均显著高于孤雌激活组(P<0.05),延边黄牛孤雌激活胚胎O2·-变化以2细胞期最高,4细胞期最低,8细胞期有所上升,但低于2细胞期水平,16细胞期下降,囊胚期又再升高。延边黄牛体细胞克隆胚胎O2·-变化以2细胞期最低,4细胞期急速上升,8细胞期降低,16细胞期开始上升,至囊胚期基本持平；各细胞期体细胞克隆胚胎H2O2水平均显著高于孤雌激活胚胎(P<0.05),延边黄牛孤雌激活胚胎H2O2变化为2细胞期至囊胚期持续升高,延边黄牛体细胞克隆胚胎H2O2变化以2细胞期最低,4细胞期急速上升,8细胞期降低后至囊胚期持续上升。(4)卵母细胞时期编码CAT＞Mn-SOD和GPx的基因均无表达,2细胞时期体细胞克隆胚胎编码CAT的基因有表达,4细胞时期体细胞克隆胚胎编码CAT和GPx的基因有表达,孤雌激活胚胎编码CAT的基因有表达,8细胞时期和16细胞以上时期体细胞克隆和孤雌激活胚胎编码CAT、Mn-SOD和GPx的基因均有表达；延边黄牛核移植和孤雌激活胚胎编码CAT的基因表达丰度之间在2细胞期以上均存在显著差异(P<0.05),延边黄牛核移植和孤雌激活胚胎编码Mn-SOD的基因表达丰度之间在8细胞期差异不显著(P>0.05),16细胞期以上时期差异显著(P<0.05),延边黄牛核移植和孤雌激活胚胎编码GPx的基因表达丰度之间在4细胞期差异显著(P<0.05),8细胞期以上时期差异不显著(P<0.05)。结论：(1)H2O2对延边黄牛体细胞克隆胚胎具有氧化损伤及加快体外发育速度的作用,GSH对延边黄牛体细胞克隆胚胎具有抗氧化保护作用；(2)延边黄牛体细胞克隆胚胎所受的氧化应激致使其氧化损伤水平高于孤雌激活胚胎,其内部ROS变化较大的时期正处于染色体基因组开始激活的时期；(3)延边黄牛体细胞克隆胚胎编码Mn-SOD和GPx的mRNA随着染色体基因组的激活而表达,大量表达的时期晚于胚胎内部ROS大量产生的时期。更多还原

【Abstract】 Yanbian cattle as one of top five local varieties cattle possess meat tender, nutrient-rich, excellent meat potential and broad prospects of products for commercial development. With the development of animal husbandry and "East West, Hong Huang," Beef Cattle Development Strategy in Jilin Province of China, the development of Yanbian cattle breeding industry has been coming faster and faster, so it is necessary to expand our industrialization scale and improve the additional value of science and technology. Somatic cell cloning technology considered as an emerging scientific and technological means, not only support embryonic stem cell technology, transgenic technology and medical and other scientific researchs, but also can be used to produce a large number of livestock in animal husbandry that have the same genotype and have a profound significance in stock breeding, but so far studies have shown that there is low efficiency of the technology produced more offspring with birth defects, etc., therefore, it does not adapt to the actual production needs. Numerous researches on oxidative damage of gametes in vitro production and storage have been reported in artificial insemination, in vitro fertilization, embryo transfer and other reproductive biotechnologies, and there have been lots of studies on micro-manipulation method, fusion activation method, the nuclear re-programming mechanism, DNA methylation etc. in the somatic cell cloning technology, now, it is a new hotspot to research the molecular mechanism of physical defects of offspring in somatic cell cloning, but the research on oxidative damage of cloned embryos of somatic cell cloning has not been reported.Objective:(1) To observe the effect of oxidant hydrogen peroxide (H2O2) on the development of cloned embryos from Yanbian cattle in vitro and explore the oxidative damage of H2O2 on the reconstructed embryos; (2) To observe the effect of anti-oxidant glutathione (GSH) on the development of cloned embryos from Yanbian cattle in vitro and explore the protective role of GSH to the oxidative damage; (3) To quantify the H2O2 and superoxide anion (O2-) two kinds of reactive oxygen species (ROS) levels within embryos cloned from Yanbian cattle in contrast to parthenogenetic embryos of Yanbian cattle and explore the change law of them at different stages of development in vitro; (4) To detect mRNA expression levels of encoding catalase (CAT), manganese super-oxide enzymes (Mn-SOD) and glutathione peroxidase (GPx) at different developmental phase of reconstructed embryos from Yanbian cattle in contrast to- parthenogenetic embryos and explore the relationship between the expression levels and oxidative damage.Methods and contents:(1) Adding exogenous H2O2 to in vitro culture medium, observe the development efficiency and development speed of reconstructed embryos from somatic cell cloning in Yanbian cattle in vitro and test the quality of the impact caused by oxidative damage to determine the critical H2O2 concentration of impact their development and the stage of impact greatest; (2) Adding exogenous GSH in vitro culture medium, observe the development efficiency of reconstructed embryos from somatic cell cloning in Yanbian cattle to determine the appropriate concentration and the required suitable time relieving oxidative damage; (3)Detect content of ROS (H2O2 and O2·- within reconstructed embryos at different developmental stages using dihydro-Ethidium and 2’,7’-dichlorofluorescein diacetate, respectively, and using fluorescent microscope camera and image processing software for quantitative analysis and explore the internal change law of ROS; (4) The mRNA of encoding CAT, Mn-SOD and GPx of reconstructed embryos were amplified using specific primers and reverse transcription-polymerase chain reaction and detected by agarose gel electrophoresis, and then determine the expression law of reconstructed embryos using gel image analysis software and semi-quantitative analysis.Results:(1) the cleavage rate of adding 0μMol/L,60μMol/L,80 uMol/LH2O2 was significantly higher than that of 100μMol/L and 120μMol/L group (P<0.05), the development rate of all of the treatment group adding H2O2 was significantly lower than the non-adding treatment group (P<0.05); the cleavage rate of the treatment group adding H2O2 at 0-24h was significantly higher than the other treatment groups (P<0.05), the development rate more than 16 cells of 48-72h treatment group was significantly lower than the other treatment groups (P<0.05); there were no significant differences in different periods of time without adding H2O2 (P> 0.05), but the development rate more than 16 cells of different periods of time non-adding H2O2 was significantly lower than that the control group (all-time do not add, P<0.05); the reconstructed embryos treated with H2O2 displayed rough blastomere surface, uneven cytoplasm, (small and loose) and occasionally saw little or no cytoplasm of the blastomere, the gap between blastomeres and zona pellucida was large, and blastomere sizes developed irregularly and differently in scale, and there were a large number of cell debris. (2) the cleavage rate of 1 mMol/L group adding exogenous GSH was significantly higher than that of 7 mMol/L group (P<0.05), the cleavage rate of 1 mMol/L and 7 mMol/L group was not significantly higher than that of 0 mMol/L and 4 mMol/L group (P> 0.05), the blastocysts rate of 1 mMol/L group was significantly higher than that of the other treatment groups (P<0.05); the cleavage rate of 0-24 h group and 24-48 h group adding exogenous GSH was significantly higher than that of 48-72 h group and 72-96h group (P<0.05), did not differ significantly with the other treatment groups (P> 0.05), the development rated of more than 16 cell of the control group was significantly higher than that of the 48-72 h group and 72-96h group (P<0.05). (3) there was no significant difference on O2-levels at 2-cell stage between parthenogenetic activation and somatic cell cloning groups (P> 0.05), the O2-levels at 4-cell stage,8-cell stage,16-cell stage and blastocyst stage of somatic cell cloning groups was significantly higher that of the parthenogenetic activation group (P<0.05), O2-level of parthenogenetic embryos from Yanbian cattle was up to the highest at 2-cell stage, down to the lowest at 4-cell stage, that of 8-cell stage has increased, decreased at 16-cell stage,and increased again at blastocyst stage. The O2-of reconstructed embryos from Yanbian cattle was lowest at 2-cell stage, increased sharply at 4-cell stage and decreased at 8-cell stage, began to rise at 16-cell stage and was basically the same to the blastocyst stage; the H2O2 level of reconstructed embryos was significantly higher than that of parthenogenetic embryos at any cell stage (P<0.05), the H2O2 level of parthenogenetic embryos continueed to rise gradually at 2-cell stage to the blastocyst stage, that of reconstructed embryos was lowest at 2-cell stage, increased at 4-cell stage sharply, reduced after the 8-cell stage and continued to rise.to the blastocyst stage. (4) there was no expression of encoding CAT, Mn-SOD and GPx genes in oocytes, there was expression of encoding CAT genes of reconstructed embryos at 2-cell stage, expression of encoding CAT and GPx genes of reconstructed embryos and encoding CAT genes of parthenogenetic embryos at 4-cell stage, expression of encoding CAT, Mn-SOD and GPx genes of reconstructed embryos and parthenogenetic embryos at 8-cell stage and more than 16-cell stage; abundance of CAT gene expression of reconstructed embryos and parthenogenetic embryos was significantly different at 2-cell stage and latter development stage (P<0.05), abundance of Mn-SOD gene expression of reconstructed embryos and parthenogenetic embryos was not significantly different at 8-cell stage (P> 0.05), but that was significantly different at 16-cell stage and latter development stage (P<0.05), abundance of GPx gene expression of reconstructed embryos and parthenogenetic embryos was significantly different at 4-cell stage, but that was not significantly different at 8-cell stage and latter development stage (P> 0.05).Conclusions:(1) H2O2 affecting on reconstructed embryos from somatic cell cloning in Yanbian cattle can result in oxidative damage to the embryos of reconstructed, and aslo accelerating the development speed. GSH has an anti-oxidant protective role to the reconstructed embryos. (2) The ROS level within the reconstructed embryos is higher than the parthenogenetic embryos, a period of great change almost focus on the chromosome genome activation period. (3) The mRNA of encoding CAT, Mn-SOD and GPx of reconstructed embryos expressed with the genomic activation and periods of expression a lot was later than the period of embryonic ROS generated within expression a lot.更多还原