延边黄牛体细胞克隆胚胎氧化损伤的研究
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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。
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.
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。
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.
引文
[1]王滇,于孟飞,张春燕,等.哺乳动物体细胞克隆的研究进展.中国畜牧兽医,2008,35(12)：110-112.
[2]Cibelli J, Steve L, Stice P, et al. Cloned transgenic calves produced from nonquiescent fetal fibroblast. Science,1998,280:1256-1258.
[3]李刚,侯文文,章孝荣,等.哺乳动物体细胞克隆技术的应用前景.中国畜牧兽医,2008,35(4)：48-51.
[4]Kato Y, Tani T, Sotomaru Y. Cloning of calves from various somatic cell types of male and female adult, newborn and fetal cows. J Repord Fertil,2000,120(2):231-237.
[5]Saltman P. Oxidative Stress:A radical view. Seminars in Hematology, 1989,26:249-256.
[6]Paolisso G, Tagliamonte MR, Rizzo MR, et al. Oxidative stress and advancing age:results in healthy centenarians. J Am Geriatr Soc, 1998,46(7):833-838.
[7]Lang CA, Naryshkin S, Schneider DL, et al. Low blood glutathione levels in healthy aging adults. J Lab Clin Med,1992,120(5):720-725.
[8]Gabby M, Tauber M, Porat S, et al. Selective role of glutathione in protecting human neuronal cells from dopamine-induced apoptosis. Neuropharmacology,1996,35(5):571-578.
[9]Nasr-esfahani MH, Aitken RJ, Johnson MH. The effect of iron and iron chelators on the in-vitro block to development of the mouse preimplantation embryo:BAT6 a new medium for improved culture of mouse embryos in vitro. Hum Reprod,1990,5(8):997-1003.
[10]Nakayama T, Noda Y, Goto Y, et al. Effects of visible light and other environmental factors on the production of oxygen radicals by hamster embryos. Theriogenology,1994,41(2):499-510.
[11]Joza N, Susin SA, Daugas E, et al. Essential role of the mitochondrial apoptosis-inducing factor programmed cell death. Nature,2001,410: 549-554.
[12]Susin SA, Lorenzo HK, Zamzami N, et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature,1999,397: 441-446.
[13]Said E1M ouatassim, Guerin P, MenzeoY. Mammalian oviduct and protection against free oxygen radicals expression of genes encoding antioxidant enzymes in human and mouse. European Journal of Obsterics,2000,89(1):1-6.
[14]Ying Peng, Pai-HaoYang, Ng Samuel SM, et al. Protection of Xenopus laevis Embryos Against Alcohol-induced Delayed Gut Maturation and Growth Retardation by Peroxiredoxin 5 and catalase. Journal ofMolecularBiology,2004,340(4):819-827.
[15]陈瑗,周玫.自由基医学基础与病理生理.北京：人民卫生出版社,2002.1-34.
[16]Mover H, Ar A. Antioxidant enzymatic activity in embryos and placenta of rats chronically exposed to hypoxia and hyperoxia. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol,1997,117 (2): 151-157
[17]Gunther T, Hollriegl V, Vormann J. Perinatal development of iron and antioxidant defence system. Trace Elem Electrolytes Health Dis,1993, 7 (1):47-52
[18]冯维杨,李俞锦,钱忠义.超氧化物歧化酶和过氧化氢酶抗葡萄糖缺失诱导的神经元损伤作用研究.昆明医学院学报,2009,(7)：11-15.
[19]Mackler B, Person RE, Nguyen TD, et al. Studies of the cellular distribution of superoxide dismutases in adult and fetal rat tissues. Free Radic Res,1998,28(2):125-129.
[20]黄勇,廖彪.超氧化物歧化酶在斑马鱼早期胚胎中的表达特点.陕西理工学院学报(自然科学版),2008,24(3)：91-94.
[21]周玫,陈瑗.自由基的产生及其在某些疾病发生中的作用.中国病理生理志,1987,(3)：176.
[22]Lee CS, Koo DB, Fang NZ, et al. Potent and stage-specific action of glutathione on the development of goat early embryos in vitro. Mol Reprod Dev,2000,57:48-54.
[23]Nasr-esfahani MH, Aitken RJ, Johnson MH. The effect of iron and iron chelators on the in-vitro block to development of the mouse preimplantation embryo:BAT6 a new medium for improved culture of mouse embryos in vitro. Hum Reprod,1990,5(8):997-1003.
[24]Nakayama T, Noda Y, Goto Y, et al. Effects of visible light and other environmental factors on the production of oxygen radicals by hamster embryos [J]. Theriogenology,1994,41(2):499-510.
[25]Tamura H, Takasaki A, Miwa I, et al. Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate. J Pineal Res,2008,44(3):280-287.
[26]Kitagawa Y, Suzuki K, Yoneda A, et al. Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos. Theriogenology,2004,62(7):1186-1197.
[27]Suzuki C, Yoshioka K, Sakatani M, et al. Glutamine and hypotaurine improves intracelluLar oxidative status and in vitro development of porcine preimplantation embryos. Zygote,2007,15(4):317-324.
[28]曹新燕,付秋华,方南洙.活性氧对哺乳动物早期胚胎体外发育阻滞的研究进展.中国畜牧兽医,2007,34(4)：47-48.
[29]Finkel T, Holbrook N J. Oxidants, oxidative stress and the biology of ageing. Nature,2000,408(6809):239-247.
[30]Goto Y, Noda Y, Mori T, et al. Increased generation of reactive oxygen species in embryos cultured in vitro. Free Radic Biol Med, 1993,15(1):69-75.
[31]Karja NW, Kikuchi K, Fahrudin M, et al. Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions[J]. Reprod Biol Endocrinol,2006,4:54.
[32]孔祥锋,陈婉蓉,倪兵.H2O2对大鼠神经细胞的氧化损伤及硒的保护作用.卫生毒理学杂志,2000,14(2)：91-94.
[33]常福厚,扈廷茂.谷胱甘肽和活性氧对哺乳动物配子作用的研究进展.动物医学进展,2005,26(12)：6-10.
[34]Lim JM, Reggio BC, Godke RA, et al. Development of in-vitro-derived bovine embryos cultured in 5%CO2 in air or in 5% O2,5%CO2 and 90%N2. Hum Reprod,1999,14(2):458-464.
[35]Edwards JL, King WA, Kawarsky SJ, et al. Responsiveness of early embryos to environmental insults:potential protective roles of HSP70 and glutathione. Theriogenology,2001,55(1):209-223.
[36]Gardner DK. Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture. Theriogenology,1998,49(1):83-102.
[37]Chatot CL, Tasca RJ, Ziomek CA. Glutamine uptake and utilization by preimplantation mouse embryos in CZB medium. J Reprod Fertil, 1990,89(1):335-346.
[38]Makino A, Skelton MM, Zou AP, et al. Increased renal meduLlary H2O2 leads to hypertension. Hypertension,2003,42(1):25-30.
[39]Gao YJ, Lee RM. Hydrogen peroxide is an endothelium-dependent contracting factor in rat renal artery. Br J Pharmacol,2005,146(8): 1061-1068.
[40]陈修平,杜冠华.二氯荧光素双醋酸盐40年的应用与争论.医药导报,2007,26(4)：343-348.
[41]秦涛余,陈志伟.机体内活性氧生理功能研究进展.生命科学仪器,2008,6(2)：12-16.
[42]孙学军.氢分子医学研究进展[R/OL]. (2009-3-11)[2009-08-30]. http://www.sciencenet.cn/upload/blog/file/2009/3/200931115362248 1590.pdf
[43]李晓霞,刁云飞,李钟淑,等.核移植胚胎体内外活性氧的产生·危害及清除.安徽农业科学,2008,36(5)：1889-1890.
[44]Guthrie HD, Welch GR. Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry. J Anim Sci,2006,84(8):2089-2100.
[45]Karja NW, Kikuchi K, Fahrudin M, et al. Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions. Reprod Biol Endocrinol,2006,4:54.
[46]Nasr-Esfahani MH, Aitken JR, Johnson MH. Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo. Development,1990,109(2):501-507
[47]Yang HW, Hwang KJ, Kwon HC, et al. Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos. Hum Reprod,1998,13(4):998-1002
[48]Carter WO, Narayanan PK, Robinson JP. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J Leukoc Biol,1994,55(2):253-258
[49]Zheng M, Storz G. Redox sensing by prokaryotic transcription factors. Biochem Pharmacol.2000,59(1):1-6.
[50]Harvey MB, Arcellana-Panlilio MY, Zhang X, et al. Expression of genes encoding antioxidant enzymes in preimplantation mouse and cow embryos and primary bovine oviduct cultures employed for embryo coculture. Biol Reprod,1995,53(3):532-540.
[51]Johnson MH, Nasr-Esfahani MH. Radical solutions and culture problems:could free oxygen radicals be responsible for the impaired development of preimplantation mammalian embryos in vitro. Bioessays,1994, (16):31-38.
[52]Telford NA, Watson AJ, Schultz GA. Transition from maternal to embryonic control in early mammalian development:a comparison of several species. Mol Reprod Dev,1990, (26):90-100.
[53]Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine, 2nd ed. Oxford:Oxford University Press; 1989.
[54]Gille JJP, Joenje H. Biological significance of oxygen toxicity:an introduction. In:Vigo-Lelfrey C (ed), Membrane Lipid Oxidation. Ann Arbor:CRC Press; 1991:3:1-32.
[55]El Mouatassim S, Guerin P, Menezo Y. Expression of genes encoding antioxidant enzymes in human and mouse oocytes during the final stages of maturation. Mol Hum Reprod,1999,5(8):720-725.
[56]Correa GA, Rumpf R, Mundim TC, et al. Oxygen tension during in vitro culture of bovine embryos:effect in production and expression of genes related to oxidative stress. Anim Reprod Sci,2008, 104(2-4):132-142.
[1]孙焕林.成纤维细胞对延边黄牛体细胞克隆效率的影响：[硕士学位论文].龙井：延边大学农学院,2009.
[2]孙焕林,方南洙,胡艳明,等.不同来源的牛皮肤成纤维细胞原代培养效果对核移植胚发育的影响.中国畜牧杂志,2009,45(19)：21-24.
[3]黄玲.延边黄牛成纤维细胞培养、冷冻保存及核移植效率的研究：[硕士学位论文].龙井：延边大学农学院,2007.
[4]郭杰.冷冻保存对延边黄牛体细胞培养及核移植效率的影响：[硕士学位论文].龙井：延边大学农学院,2008.
[5]苗婷婷.不同成熟因素对延边黄牛卵母细胞体外成熟及核移植后胚胎早期发育的影响：[硕士学位论文].龙井：延边大学农学院,2007.
[6]毛晶.延边黄牛体细胞克隆无血清成熟液的研究：[硕士学位论文].龙井：延边大学农学院,2008.
[7]毛晶,李钟淑,蔡文莲,等.聚乙烯醇、聚乙烯比咯烷酮和牛血清白蛋白对牛卵母细胞成熟及体细胞克隆胚胎发育的影响.黑龙江畜牧兽医,2008,(10)：12-15.
[8]胡艳明.激素对牛卵母细胞体外成熟及重组胚发育的影响：[硕士学位论文].龙井：延边大学农学院,2009.
[9]胡艳明,祖晶,孙焕林,等.FSH和LH对延边黄牛卵母细胞体外成熟及重组胚发育的影响.中国畜牧杂志,2009,45(11)：18-22.
[10]祖晶.核成熟抑制剂对牛卵母细胞体外成熟及重组胚发育的影响：[硕士学位论文].龙井：延边大学农学院,2009.
[11]祖晶,蒋涛,杨月春,等.不同核成熟抑制剂对延边黄牛卵母细胞发育的影响.江苏农业科学,2009,(4)：233-236.
[12]李井春.延边黄牛体细胞克隆卵母细胞显微操作方法的研究：[硕士学位论文].龙井：延边大学农学院,2007.
[13]李井春,方南洙.延边黄牛卵母细胞去核方法的研究.中国牛业科学,2006,32(5)：19-22.
[14]王艳多.化学诱导去核法在延边黄牛体细胞核移植中的应用研究：[硕士学位论文].龙井：延边大学农学院,2008.
[15]李雪峰,安志兴,李向臣,等.紫外线照射对牛孤雌激活卵母细胞和体细胞核移植胚胎体外发育的影响。中国兽医科技,2003,33(3)： 20-25.
[16]于莹.荧光辅助去核在延边黄牛体细胞核移植中的应用：[硕士学位论文].龙井：延边大学农学院,2009.
[17]林涛.末期去核在延边黄牛体细胞核移植中的应用研究：[硕士学位论文].龙井：延边大学农学院,2009.
[18]林涛,李钟淑,方南洙.改进的末期去核方法解决延边黄牛卵母细胞第一极体残留问题.江苏农业科学,2009,(5)：198-200.
[19]林涛,方南洙,宋君博,等.挤压去核法和盲吸去核法在延边黄牛体细胞核移植中的对比研究.江苏农业科学,2009(2)：178-180.
[20]林涛,胡艳明,祖晶,等.末期去核在牛体细胞核移植中的应用.江苏农业科学,2009,(3)：247-248.
[21]林涛,李晓霞,刁云飞,等.延边黄牛卵母细胞激活后第二极体排出的研究.畜牧与兽医,2008,40(8)：50-52.
[22]刁云飞.延边黄牛体细胞克隆显微操作液的研究：[硕士学位论文].龙井：延边大学农学院,2008.
[23]刁云飞,李晓霞,李钟淑,等.显微操作液中添加抗氧化剂对延边黄牛体细胞克隆效率的影响.畜牧与兽医,2008,40(6)：57-59.
[24]杨月春.延边黄牛体细胞克隆融合、激活条件的研究：[硕士学位论文].龙井：延边大学农学院,2007.
[25]杨月春,李钟淑,金一,等.延边黄牛体细胞克隆激活条件的研究.延 边大学农学学报,2008,30(1)：26-30.
[26]杨月春,李钟淑,王士勇,等.延边黄牛体细胞克隆融合条件的研究.延边大学农学学报,2009,31(3)：153-158.
[27]李晓霞.延边黄牛体细胞克隆电融合的研究：[硕士学位论文].龙井：延边大学农学院,2008.
[28]李晓霞,刁云飞,李钟淑,等.不同融合液对延边黄牛体细胞克隆核移植效率的影响.安徽农业科学,2008,36(4)：1454-1456.
[29]李晓霞,刁云飞,李钟淑,等.融合液浓度梯度融合法不同融合条件对延边黄牛体细胞克隆效率的影响.畜牧与兽医,2009,41(2)：45-47.
[30]李晓霞,刁云飞,李钟淑,等.融合液浓度梯度融合法提高延边黄牛体细胞克隆效率.中国畜牧杂志,2008,44(13)：19-21.
[31]宋君博.细胞松弛素B和咖啡因对延边黄牛体细胞克隆效率的影响：[硕士学位论文].龙井：延边大学农学院,2009.
[32]宋君博,林涛,胡艳明,等.细胞松弛素B对延边黄牛体细胞核移植效率的影响.河南农业科学,2009,(4)：123-126.
[33]王士勇.不同培养体系对延边黄牛体细胞克隆胚胎体外发育的影响：[硕士学位论文].龙井：延边大学农学院,2007.
[34]王士勇,李钟淑,金庆国,等.共培养对延边黄牛重组胚体外发育的影响.延边大学农学学报,2008,30(3)：192-196.
[35]韩岩,尹多,林涛,等.氨基酸对延边黄牛体细胞克隆重组胚发育的影响.见：东北三省及内蒙古地区遗传学研究进展学术研讨会论文汇编.哈尔滨：哈尔滨医科大学,2009.
[36]尹多,林涛,韩岩,等.抗氧化剂对牛重组胚发育的影响.见：东北三省及内蒙古地区遗传学研究进展学术研讨会论文汇编.哈尔滨：哈尔滨医科大学,2009.
[37]Eggan K, Akutsu H, Hochedlinger K, et al. X-Chromosome inactivation in cloned mouse embryos. Science,2000,290(5496): 1578-1581.
[38]Clerc P, Avner P. Reprogramming X inactivation. Science,2000, 290(5496):1518-1519.
[39]Smith SL, Everts RE, Tian XC, et al. Global gene expression profiles reveal significant nuclear reprogramming by the blastocyst stage after cloning. Proc Natl Acad Sci USA,2005,102(49):17582-17587.
[40]Kubota C, Yamakuchi H, Todoroki J, et al. Six cloned calves produced from adult fibroblast cells after long-term culture. Proc Natl Acad Sci USA,2000,97(3):990-995.
[41]Wakayama T, Tabar V, Rodriguez I, et al. Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science,2001,292(5517):740-743.
[42]Wrenzycki C, Lucas-Hahn A, Herrmann D, et al. In vitro production and nuclear transfer affect dosage compensation of the X-linked gene transcripts G6PD, PGK, and Xist in preimplantation bovine embryos. Biol Reprod,2002,66(1):127-134.
[43]吴昊.延边黄牛体细胞克隆胚胎的核型分析：[硕士学位论文].龙井：延边大学农学院,2009.
[44]Abe H, Yamashita S, Satoh T, et al. Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serumfree or serumcontaining media. Mol Reprod Dev,2002,61:57-66.
[45]敬晓棋.山羊早期胚胎端粒酶活性测定及克隆超微结构观察：[硕士学位论文].陕西咸阳：西北农林科技大学,2005.
[46]张晓红,方南洙,崔成都.延边黄牛卵母细胞于体细胞克隆胚胎内脂滴变化规律的研究.畜牧与兽医,2008,40(10)：10-13.
[47]张晓红,崔成都,方南洙,等.延边黄牛体细胞克隆早期胚胎部分超微机构的研究.畜牧与兽医,2009,41(9)：45-48.
[1]Burdon RH. Superoxide and hydrogen peroxide in relation to mammalian cell proliferation. Free Radie Biol Med,1995,18(4): 775-794.
[2]Hunt CR, Sim JE, Sullivan SJ, et al. Genomic instability and catalase gene amplification induced by chronic exposure to oxidative stress.Cancer Res,1998,58(14):3986-3992.
[3]Morel F, Cohen Tanugi Cholley L, Brandolin G et al. The O2" generating oxidase of B lymphocytes:Epstein-Barr virus-immortalized B lymphocytes as a tool for the identification of defective components of the oxidase in chronic granulomatous disease. Biochiem Biophys Acta,1993,1182(1):101-109.
[4]Forsberg L, de Faire U, Morgenstern R. Oxidative stress, human genetic variation, and disease. Arch Biochem Biophys,2001,389(1): 84-93.
[5]Gunther T, Hollriegl V, Vormann J. Perinatal development of iron and antioxidant defence system. Trace Elem Electrolytes Health Dis,1993, 7(1):47-52
[6]Mackler B, Person RE, Nguyen TD, et al. Studies of the cellular distribution of superoxide dismutases in adult and fetal rat tissues. Free Radic Res,1998,28(2):125-129.
[7]Mover H, Ar A. Antioxidant enzymatic activity in embryos and placenta of rats chronically exposed to hypoxia and hyperoxia. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol,1997,117 (2): 151-157.
[8]Finkel T. Oxygen radicals and signaling. Curr Opin Cell Biol,1998, 10(2):248-253.
[9]Finkel T, Holbrook NJ. Holbrook. Oxidants, oxidative stress and the biology of ageing. Nature,2000,408(6809):239-247.
[10]Saltman P. Oxidative Stress:A radical view. Seminars in Hematology, 1989,26:249-256.
[11]Albina JE, Cui S, Mateo RB, et al. Nitric oxide-mediated apoptosis in murine peritoneal macrophages. J Immunol,1993,150(11): 5080-5085.
[12]周彦娇.活性氧在线粒体凋亡信号传导中的调节作用.中国运动医学杂志,2005,24(6)：746-750.
[13]Keizer HG, Pinedo HM, Schaurhuis GJ, et al. Doxorubicin (adriamycin):a critical review of free radical dependent mechanisms of cytotoxicity. Pharmacol Ther,1990,47:219-231.
[14]Bickers DR, Das M, Mukhtar H, et al. Photoenhancement of lipid peroxidation associated with the generation of reactive oxygen species in hepatic microsomes of hematoporphyrin derivative-treated rats. Cancer Res,1985,45:6328-6330.
[15]Tang FJ, Ts'o PO, Lesko SA. Simultaneous in situ detection of beta-interferon mRNA and DNA in the same cell. J Histochem Cytochem,1989,37:697-701.
[16]Fujitani Y, Kasai K, Ohtani S, et al. Effect of oxygen concentration and free radicals on in vitro development of in vitro-produced bovine embryos. J Anim Sci,1997,75:483-489.
[17]Takahashi M, Keicho K, Takahashi H, et al. Effect of oxidative stress on development and DNA damage in in-vitro cultured bovine embryos by comet assay. Theriogenology,2000,54:137-145.
[18]Hashimoto S, Minami N, Takakura R, et al. Low oxygen tension during in vitro maturation is beneficial for supporting the subsequent development of bovine cumulus-oocyte complexes. Mol Reprod Dev, 2000,57:353-360.
[19]Mizushima S, Fukui Y. Fertilizability and developmental capacity of bovine oocytes cultured individually in a chemically defined maturation medium. Theriogenology,2001,55:1431-1445.
[20]de Matos DG, Furnus CC. The importance of having high glutathione (GSH) level after bovine in vitro maturation on embryo development effect of beta-mercaptoethanol, cysteine and cystine. Theriogenology, 2000,53:761-771.
[21]布拉舍.J.发育的生物化学.严绍颐,译.北京：科学出版社,1964,164-224.
[22]郑荣梁,黄中洋.自由基医学与农学基础.北京：高等教育出版社,2001,1-14,68-79,113-123.
[23]Moskovitz J. Methionine sulfoxide reductases ubiquitous enzymes involved in antioxidant defense, protein regulation, and prevention of aging-associated diseases. Biochim Biophys Acta,2005,1703(2): 213-219.
[24]Said ElMouatassim, Guerin P, Menzeo Y. Mammalian oviduct and protection against free oxygen radicals expression of genes encoding antioxidant enzymes in human and mouse. European Journal of Obsterixs,2000,89(1):1-6.
[25]Peng Y, Yang PH, Ng SS, et al. Protection of Xenopus laevis Embryos Against Alcohol-induced Delayed Gut Maturation and Growth Retardation by Peroxiredoxin 5 and catalase. Journal of Molecular Biology,2004,340(4):819-827.
[26]陈瑗,周玫.自由基医学基础与病理生理.北京：人民卫生出版社,2002：1-34.
[27]周玫,陈瑗.自由基的产生及其在某些疾病发生中的作用.中国病理生理杂志,1987,3：176.
[28]Lee CS, Koo DB, Fang NZ, et al. Potent and stage-specific action of glutathione on the development of goat early embryos in vitro. Mol Reprod Dev,2000,57:48-54.
[29]Ikegami T, Suzuki Y, Shimizu T. et al. Model mice for tissue-specific deletion of the manganese superoxide dism utase (MnSOD) gene. Biochem Biophys Res Commun,2002,296:729-736.
[30]Marklund SL, Holme E, Hellner L. Supemxide dismutase in extracellular fluids. Clin Chim Acta,1982,126:41-51.
[31]Mannick JB, Asano K, lzumi K, et al. Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell,1994,79:1137-1146.
[32]Devi M, Das N. Antiproliferative effect of polyunsaturated fatty acids and interleukin-2 on normal and abnormal human lymphocytes. Experientia,1994,50(5):489-492.
[33]Yan Oberley LW, Zhong W, et al. Manganese-containing superoxide dismutase overexpression causes phenotypic reversion in SV40-transformed human lung fibroblasts. Cancer Res,1996,56(12): 2864-2971.
[34]Storz G, Tartaglia LA, Ames BN. Transcriptional regulator of oxidative stress-inducible genes:direct activation by oxidation. Science,1990,248(4592):189-194.
[35]Yumin Hu, Daniel G, Rosen, Yan Zhou, et al. Mitochondrial MnSOD Expression in Ovarian Cancer:Role in Cell Proliferation and Response to Oxidative Stress. JBC,2005,9:1-16.
[36]Jones SP, Hoffmeyer MR, Sharp BR, et al. Role of intracellular antioxidant enzymes after in vivo myocardial ischemia and reperfusion. Am J Physiol Heart Circ Physiol,2003,284:H277-H282.
[37]Yan C, Huang A, Wu Z, et al. Increased superoxide leads to decreased flow-induced dilation in resistance arteries of Mn-SOD-deficient mice. Am J Physiol Heart Circ Physiol,2005,288:H2225-H2231.
[38]Qi X, Lewin AS, Hauswirth WW, et al. Optic neuropathy induced by reductions in mitochondrial superoxide dismutase. Invest Ophthalmol Vis Sci,2003,44:1088-1096.
[39]Qi X, Lewin AS, Sun L, et al. SOD gene transfer protects against optic neuropathy induced by deficiency of complex I. Ann Neurol, 2004,56:182-191.
[40]Strassburger M, Bloch W, Sulyok S, et al. Heterozygous deficiency of manganese superoxide dismutase results in severe lipid peroxidation and spontaneous apoptosis in murine myocardium in vivo. Free Radic Biol Med,2005,38:1458-1470.
[41]刘永彪,刘颖,赵杰.SOD对肿瘤细胞凋亡影响和抗氧化损伤的研究：Ⅰ.SOD与DDP、ADM、VP16合用对肿瘤细胞凋亡的影响.辐射研究与辐射工艺学报,2004,22(2)：114-122.
[42]刘永彪,刘颖,赵杰.SOD对肿瘤细胞凋亡影响和抗氧化损伤的研究：Ⅱ.SOD对荷瘤小鼠脾淋巴细胞的辐射防护效应.辐射研究与辐射工艺学报,2004,22(3)：165-170.
[43]黄勇,廖彪.超氧化物歧化酶在斑马鱼早期胚胎中的表达特点.陕西理工学院学报(自然科学版),2008,24(3)：91-94.
[44]Yuan HT, Bingle CD, Kelly FJ. Differential patterns of antioxidant enzyme mRNA expression in guinea pig lung and liver during development. Biochim Biophys Acta,1996,1305(3):163-171.
[45]Gomez-Anduro GA, Barillas-Mury CV, Peregrino-Urate AB, et al. The cytosolic manganese superoxide dismutase from the shrimp Litopenaeus vannamei:Molecular cloning an expression. Dev Comp Immunol,2006,30(10):893-900.
[46]高永刚.壬基酚对水生生物抗氧化酶活性的影响：[硕士学位论文].山东青岛：中国海洋大学,2006.
[47]王会文,刘建荣,静天玉,等.微量超氧化物歧化酶邻苯三酚／Vc测活法.河北大学学报(自然科学版),2000.20(1)：61-62.
[48]Schrader M, Fahimi HD. Peroxisomes and oxidative stress. Biochimica et Biophysica Acta (BBA),2006,1763(12):1755-1766.
[49]Zamocky M, Furtmuller PG, Obin C. Evolution of catalases from bacteria to humans. Antioxidants & Redox Signaling,2008,10(9): 1527-1548.
[50]张坤生,田荟琳.过氧化氢酶的功能及研究.食品科技,2007,1：8-11.
[51]Pinsent HD. Production and utilization of catalase using Saccharomyces cerevisiae. Biochem,1948,43:193-203.
[52]Summer JB, Dounce AL. Purification and characterization of a thermostable catalase from culture broth of Thermoascus aurantiacus. J Biol Chem,1937,121:417-424.
[53]Goth L. Production of catalase and glucose oxidase by Aspergillus niger using unconventional oxygenation of culture. Clin Chim Acta, 1991,196:143.
[54]Kono Y, Fridovich I. Isolation and characterization of the pseudo-catalase of Lactobacillus plantarum-A new manganese containing enzyme. J Biol Chem,1983,258(10):6015-6019.
[55]Barynin VV, Grebenko AI. T-catalase is a nonheme catalase of extremely thermophilic bacterium Thermusthermophilus. Dokl Akad Nauk SSSR,1986,286(2):461-464.
[56]赵保路.氧自由基和天然抗氧化剂.北京：科学出版社,1999.
[57]张波,刘承芸,孟紫强.二氧化硫吸入对小鼠各脏器过氧化氢酶活性的影响.环境与职业医学,2003,20(1)：10-12.
[58]冯维杨,李俞锦,钱忠义.超氧化物歧化酶和过氧化氢酶抗葡萄糖缺失诱导的神经元损伤作用研究.昆明医学学报,2009,(7)：11-15.
[59]Glenn S, Gerhard, Elizabeth J, et al. Molecular cloning and sequence analysis of theDanio reriocatalase gene. Comp Biochem Physiol B Biochem Mol Biol,2000,127:447-457.
[60]Chen X, Liang H, Remmen HV, et al. Catalase transgenic mice: characterization and sensitivity to oxidative stress. Archives of Biochemistry and Biophysics,2004,422:197-210.
[61]Gerhard GS, Kauffman EJ, Grundy MA. Molecular cloning and sequence analysis of theDanio reriocatalase gene.Comp Biochem Physiol B Biochem Mol Biol,2000,127(4):447-457.
[62]雷厚成,李毓琦,魏素萱,等.过氧化氢酶活性的化学发光法测定.华西药学杂志,1994,9(3)：176-178.
[63]徐镜波,郎佩珍.过氧化氢酶活性及活性抑制的测定.环境化学,1994,13(3)：279-282.
[64]彭志英,蒋黎.紫外速率直接法测定过氧化氢酶活性.华西医学,1995,10(1)：4-7.
[65]陈大义.催化分光光度法快速测定血清过氧化氢酶活性.四川省卫生管理干部学院学报,2000,19(4)：249-251.
[66]周强,曹春艳.血清过氧化氢酶的比色测定.哈尔滨医科大学学报。2001,35(6)：473-474.
[67]禹邦超,刘德立.应用酶学导论.武汉：华中师范大学出版社,1994,12 (1).
[68]董泗建,刘昌玲.一种鉴定过氧化氢酶活性的铁染色法.生物化学与生物物理进展,1996,23(1)：86-88.
[69]薛颖,刘平,刘泰然,等.酶-氧电极测定固定食品中H2O2方法的研究.卫生研究,2006,35(3)：313-316.
[70]姜招峰,周翔.血清过氧化氢酶活性快速测定的新方法—极谱氧电极法.白求恩医科大学学报,1989,15(5)：464-467.
[71]潘建国,王开发,郑尧隆,等.用Warburg法测定蜂花粉中过氧化氢酶活性.植物学报,2001,18(4)：505-508.
[72]罗泽娇,张随成.简易气量法测定土壤过氧化氢酶活性的研究.地质科技情报,2005,24(4)：87-90.
[73]陈瑷,周玫.生命的化学.1983；3(6)：16.
[74]蔡晓波,陆伦根.谷胱甘肽过氧化物酶与肝脏疾病.世界华人消化杂志,2009,17(32)：3279-3282.
[75]苗雨辰,白玲,苗琛,等.植物谷胱甘肽过氧化物酶研究进展.植物学通报,2005,22(3)：350-356.
[76]张舸,田东萍,苏敏,等.长期低硒、低碘对发育期大鼠肝脏和全血GSH-Px活性的影响.环境与职业医,2005,22：309-311.
[77]孙娟,陈瑗,周玫.GPx对活性氧诱发细胞程序性死亡的影响.生物化学与生物物理进展,1998,25(3)：259-262.
[78]Fukuhara R, Kageyama T. Tissue distribution, molecular cloning, and gene expression of cytosolic glutathione peroxidase in Japanese monkey. Zoolog Sci,2003,20(7):861-868.
[2]Cibelli J, Steve L, Stice P, et al. Cloned transgenic calves produced from nonquiescent fetal fibroblast. Science,1998,280:1256-1258.
[3]李刚,侯文文,章孝荣,等.哺乳动物体细胞克隆技术的应用前景.中国畜牧兽医,2008,35(4)：48-51.
[4]Kato Y, Tani T, Sotomaru Y. Cloning of calves from various somatic cell types of male and female adult, newborn and fetal cows. J Repord Fertil,2000,120(2):231-237.
[5]Saltman P. Oxidative Stress:A radical view. Seminars in Hematology, 1989,26:249-256.
[6]Paolisso G, Tagliamonte MR, Rizzo MR, et al. Oxidative stress and advancing age:results in healthy centenarians. J Am Geriatr Soc, 1998,46(7):833-838.
[7]Lang CA, Naryshkin S, Schneider DL, et al. Low blood glutathione levels in healthy aging adults. J Lab Clin Med,1992,120(5):720-725.
[8]Gabby M, Tauber M, Porat S, et al. Selective role of glutathione in protecting human neuronal cells from dopamine-induced apoptosis. Neuropharmacology,1996,35(5):571-578.
[9]Nasr-esfahani MH, Aitken RJ, Johnson MH. The effect of iron and iron chelators on the in-vitro block to development of the mouse preimplantation embryo:BAT6 a new medium for improved culture of mouse embryos in vitro. Hum Reprod,1990,5(8):997-1003.
[10]Nakayama T, Noda Y, Goto Y, et al. Effects of visible light and other environmental factors on the production of oxygen radicals by hamster embryos. Theriogenology,1994,41(2):499-510.
[11]Joza N, Susin SA, Daugas E, et al. Essential role of the mitochondrial apoptosis-inducing factor programmed cell death. Nature,2001,410: 549-554.
[12]Susin SA, Lorenzo HK, Zamzami N, et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature,1999,397: 441-446.
[13]Said E1M ouatassim, Guerin P, MenzeoY. Mammalian oviduct and protection against free oxygen radicals expression of genes encoding antioxidant enzymes in human and mouse. European Journal of Obsterics,2000,89(1):1-6.
[14]Ying Peng, Pai-HaoYang, Ng Samuel SM, et al. Protection of Xenopus laevis Embryos Against Alcohol-induced Delayed Gut Maturation and Growth Retardation by Peroxiredoxin 5 and catalase. Journal ofMolecularBiology,2004,340(4):819-827.
[15]陈瑗,周玫.自由基医学基础与病理生理.北京：人民卫生出版社,2002.1-34.
[16]Mover H, Ar A. Antioxidant enzymatic activity in embryos and placenta of rats chronically exposed to hypoxia and hyperoxia. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol,1997,117 (2): 151-157
[17]Gunther T, Hollriegl V, Vormann J. Perinatal development of iron and antioxidant defence system. Trace Elem Electrolytes Health Dis,1993, 7 (1):47-52
[18]冯维杨,李俞锦,钱忠义.超氧化物歧化酶和过氧化氢酶抗葡萄糖缺失诱导的神经元损伤作用研究.昆明医学院学报,2009,(7)：11-15.
[19]Mackler B, Person RE, Nguyen TD, et al. Studies of the cellular distribution of superoxide dismutases in adult and fetal rat tissues. Free Radic Res,1998,28(2):125-129.
[20]黄勇,廖彪.超氧化物歧化酶在斑马鱼早期胚胎中的表达特点.陕西理工学院学报(自然科学版),2008,24(3)：91-94.
[21]周玫,陈瑗.自由基的产生及其在某些疾病发生中的作用.中国病理生理志,1987,(3)：176.
[22]Lee CS, Koo DB, Fang NZ, et al. Potent and stage-specific action of glutathione on the development of goat early embryos in vitro. Mol Reprod Dev,2000,57:48-54.
[23]Nasr-esfahani MH, Aitken RJ, Johnson MH. The effect of iron and iron chelators on the in-vitro block to development of the mouse preimplantation embryo:BAT6 a new medium for improved culture of mouse embryos in vitro. Hum Reprod,1990,5(8):997-1003.
[24]Nakayama T, Noda Y, Goto Y, et al. Effects of visible light and other environmental factors on the production of oxygen radicals by hamster embryos [J]. Theriogenology,1994,41(2):499-510.
[25]Tamura H, Takasaki A, Miwa I, et al. Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate. J Pineal Res,2008,44(3):280-287.
[26]Kitagawa Y, Suzuki K, Yoneda A, et al. Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos. Theriogenology,2004,62(7):1186-1197.
[27]Suzuki C, Yoshioka K, Sakatani M, et al. Glutamine and hypotaurine improves intracelluLar oxidative status and in vitro development of porcine preimplantation embryos. Zygote,2007,15(4):317-324.
[28]曹新燕,付秋华,方南洙.活性氧对哺乳动物早期胚胎体外发育阻滞的研究进展.中国畜牧兽医,2007,34(4)：47-48.
[29]Finkel T, Holbrook N J. Oxidants, oxidative stress and the biology of ageing. Nature,2000,408(6809):239-247.
[30]Goto Y, Noda Y, Mori T, et al. Increased generation of reactive oxygen species in embryos cultured in vitro. Free Radic Biol Med, 1993,15(1):69-75.
[31]Karja NW, Kikuchi K, Fahrudin M, et al. Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions[J]. Reprod Biol Endocrinol,2006,4:54.
[32]孔祥锋,陈婉蓉,倪兵.H2O2对大鼠神经细胞的氧化损伤及硒的保护作用.卫生毒理学杂志,2000,14(2)：91-94.
[33]常福厚,扈廷茂.谷胱甘肽和活性氧对哺乳动物配子作用的研究进展.动物医学进展,2005,26(12)：6-10.
[34]Lim JM, Reggio BC, Godke RA, et al. Development of in-vitro-derived bovine embryos cultured in 5%CO2 in air or in 5% O2,5%CO2 and 90%N2. Hum Reprod,1999,14(2):458-464.
[35]Edwards JL, King WA, Kawarsky SJ, et al. Responsiveness of early embryos to environmental insults:potential protective roles of HSP70 and glutathione. Theriogenology,2001,55(1):209-223.
[36]Gardner DK. Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture. Theriogenology,1998,49(1):83-102.
[37]Chatot CL, Tasca RJ, Ziomek CA. Glutamine uptake and utilization by preimplantation mouse embryos in CZB medium. J Reprod Fertil, 1990,89(1):335-346.
[38]Makino A, Skelton MM, Zou AP, et al. Increased renal meduLlary H2O2 leads to hypertension. Hypertension,2003,42(1):25-30.
[39]Gao YJ, Lee RM. Hydrogen peroxide is an endothelium-dependent contracting factor in rat renal artery. Br J Pharmacol,2005,146(8): 1061-1068.
[40]陈修平,杜冠华.二氯荧光素双醋酸盐40年的应用与争论.医药导报,2007,26(4)：343-348.
[41]秦涛余,陈志伟.机体内活性氧生理功能研究进展.生命科学仪器,2008,6(2)：12-16.
[42]孙学军.氢分子医学研究进展[R/OL]. (2009-3-11)[2009-08-30]. http://www.sciencenet.cn/upload/blog/file/2009/3/200931115362248 1590.pdf
[43]李晓霞,刁云飞,李钟淑,等.核移植胚胎体内外活性氧的产生·危害及清除.安徽农业科学,2008,36(5)：1889-1890.
[44]Guthrie HD, Welch GR. Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry. J Anim Sci,2006,84(8):2089-2100.
[45]Karja NW, Kikuchi K, Fahrudin M, et al. Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions. Reprod Biol Endocrinol,2006,4:54.
[46]Nasr-Esfahani MH, Aitken JR, Johnson MH. Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo. Development,1990,109(2):501-507
[47]Yang HW, Hwang KJ, Kwon HC, et al. Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos. Hum Reprod,1998,13(4):998-1002
[48]Carter WO, Narayanan PK, Robinson JP. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J Leukoc Biol,1994,55(2):253-258
[49]Zheng M, Storz G. Redox sensing by prokaryotic transcription factors. Biochem Pharmacol.2000,59(1):1-6.
[50]Harvey MB, Arcellana-Panlilio MY, Zhang X, et al. Expression of genes encoding antioxidant enzymes in preimplantation mouse and cow embryos and primary bovine oviduct cultures employed for embryo coculture. Biol Reprod,1995,53(3):532-540.
[51]Johnson MH, Nasr-Esfahani MH. Radical solutions and culture problems:could free oxygen radicals be responsible for the impaired development of preimplantation mammalian embryos in vitro. Bioessays,1994, (16):31-38.
[52]Telford NA, Watson AJ, Schultz GA. Transition from maternal to embryonic control in early mammalian development:a comparison of several species. Mol Reprod Dev,1990, (26):90-100.
[53]Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine, 2nd ed. Oxford:Oxford University Press; 1989.
[54]Gille JJP, Joenje H. Biological significance of oxygen toxicity:an introduction. In:Vigo-Lelfrey C (ed), Membrane Lipid Oxidation. Ann Arbor:CRC Press; 1991:3:1-32.
[55]El Mouatassim S, Guerin P, Menezo Y. Expression of genes encoding antioxidant enzymes in human and mouse oocytes during the final stages of maturation. Mol Hum Reprod,1999,5(8):720-725.
[56]Correa GA, Rumpf R, Mundim TC, et al. Oxygen tension during in vitro culture of bovine embryos:effect in production and expression of genes related to oxidative stress. Anim Reprod Sci,2008, 104(2-4):132-142.
[1]孙焕林.成纤维细胞对延边黄牛体细胞克隆效率的影响：[硕士学位论文].龙井：延边大学农学院,2009.
[2]孙焕林,方南洙,胡艳明,等.不同来源的牛皮肤成纤维细胞原代培养效果对核移植胚发育的影响.中国畜牧杂志,2009,45(19)：21-24.
[3]黄玲.延边黄牛成纤维细胞培养、冷冻保存及核移植效率的研究：[硕士学位论文].龙井：延边大学农学院,2007.
[4]郭杰.冷冻保存对延边黄牛体细胞培养及核移植效率的影响：[硕士学位论文].龙井：延边大学农学院,2008.
[5]苗婷婷.不同成熟因素对延边黄牛卵母细胞体外成熟及核移植后胚胎早期发育的影响：[硕士学位论文].龙井：延边大学农学院,2007.
[6]毛晶.延边黄牛体细胞克隆无血清成熟液的研究：[硕士学位论文].龙井：延边大学农学院,2008.
[7]毛晶,李钟淑,蔡文莲,等.聚乙烯醇、聚乙烯比咯烷酮和牛血清白蛋白对牛卵母细胞成熟及体细胞克隆胚胎发育的影响.黑龙江畜牧兽医,2008,(10)：12-15.
[8]胡艳明.激素对牛卵母细胞体外成熟及重组胚发育的影响：[硕士学位论文].龙井：延边大学农学院,2009.
[9]胡艳明,祖晶,孙焕林,等.FSH和LH对延边黄牛卵母细胞体外成熟及重组胚发育的影响.中国畜牧杂志,2009,45(11)：18-22.
[10]祖晶.核成熟抑制剂对牛卵母细胞体外成熟及重组胚发育的影响：[硕士学位论文].龙井：延边大学农学院,2009.
[11]祖晶,蒋涛,杨月春,等.不同核成熟抑制剂对延边黄牛卵母细胞发育的影响.江苏农业科学,2009,(4)：233-236.
[12]李井春.延边黄牛体细胞克隆卵母细胞显微操作方法的研究：[硕士学位论文].龙井：延边大学农学院,2007.
[13]李井春,方南洙.延边黄牛卵母细胞去核方法的研究.中国牛业科学,2006,32(5)：19-22.
[14]王艳多.化学诱导去核法在延边黄牛体细胞核移植中的应用研究：[硕士学位论文].龙井：延边大学农学院,2008.
[15]李雪峰,安志兴,李向臣,等.紫外线照射对牛孤雌激活卵母细胞和体细胞核移植胚胎体外发育的影响。中国兽医科技,2003,33(3)： 20-25.
[16]于莹.荧光辅助去核在延边黄牛体细胞核移植中的应用：[硕士学位论文].龙井：延边大学农学院,2009.
[17]林涛.末期去核在延边黄牛体细胞核移植中的应用研究：[硕士学位论文].龙井：延边大学农学院,2009.
[18]林涛,李钟淑,方南洙.改进的末期去核方法解决延边黄牛卵母细胞第一极体残留问题.江苏农业科学,2009,(5)：198-200.
[19]林涛,方南洙,宋君博,等.挤压去核法和盲吸去核法在延边黄牛体细胞核移植中的对比研究.江苏农业科学,2009(2)：178-180.
[20]林涛,胡艳明,祖晶,等.末期去核在牛体细胞核移植中的应用.江苏农业科学,2009,(3)：247-248.
[21]林涛,李晓霞,刁云飞,等.延边黄牛卵母细胞激活后第二极体排出的研究.畜牧与兽医,2008,40(8)：50-52.
[22]刁云飞.延边黄牛体细胞克隆显微操作液的研究：[硕士学位论文].龙井：延边大学农学院,2008.
[23]刁云飞,李晓霞,李钟淑,等.显微操作液中添加抗氧化剂对延边黄牛体细胞克隆效率的影响.畜牧与兽医,2008,40(6)：57-59.
[24]杨月春.延边黄牛体细胞克隆融合、激活条件的研究：[硕士学位论文].龙井：延边大学农学院,2007.
[25]杨月春,李钟淑,金一,等.延边黄牛体细胞克隆激活条件的研究.延 边大学农学学报,2008,30(1)：26-30.
[26]杨月春,李钟淑,王士勇,等.延边黄牛体细胞克隆融合条件的研究.延边大学农学学报,2009,31(3)：153-158.
[27]李晓霞.延边黄牛体细胞克隆电融合的研究：[硕士学位论文].龙井：延边大学农学院,2008.
[28]李晓霞,刁云飞,李钟淑,等.不同融合液对延边黄牛体细胞克隆核移植效率的影响.安徽农业科学,2008,36(4)：1454-1456.
[29]李晓霞,刁云飞,李钟淑,等.融合液浓度梯度融合法不同融合条件对延边黄牛体细胞克隆效率的影响.畜牧与兽医,2009,41(2)：45-47.
[30]李晓霞,刁云飞,李钟淑,等.融合液浓度梯度融合法提高延边黄牛体细胞克隆效率.中国畜牧杂志,2008,44(13)：19-21.
[31]宋君博.细胞松弛素B和咖啡因对延边黄牛体细胞克隆效率的影响：[硕士学位论文].龙井：延边大学农学院,2009.
[32]宋君博,林涛,胡艳明,等.细胞松弛素B对延边黄牛体细胞核移植效率的影响.河南农业科学,2009,(4)：123-126.
[33]王士勇.不同培养体系对延边黄牛体细胞克隆胚胎体外发育的影响：[硕士学位论文].龙井：延边大学农学院,2007.
[34]王士勇,李钟淑,金庆国,等.共培养对延边黄牛重组胚体外发育的影响.延边大学农学学报,2008,30(3)：192-196.
[35]韩岩,尹多,林涛,等.氨基酸对延边黄牛体细胞克隆重组胚发育的影响.见：东北三省及内蒙古地区遗传学研究进展学术研讨会论文汇编.哈尔滨：哈尔滨医科大学,2009.
[36]尹多,林涛,韩岩,等.抗氧化剂对牛重组胚发育的影响.见：东北三省及内蒙古地区遗传学研究进展学术研讨会论文汇编.哈尔滨：哈尔滨医科大学,2009.
[37]Eggan K, Akutsu H, Hochedlinger K, et al. X-Chromosome inactivation in cloned mouse embryos. Science,2000,290(5496): 1578-1581.
[38]Clerc P, Avner P. Reprogramming X inactivation. Science,2000, 290(5496):1518-1519.
[39]Smith SL, Everts RE, Tian XC, et al. Global gene expression profiles reveal significant nuclear reprogramming by the blastocyst stage after cloning. Proc Natl Acad Sci USA,2005,102(49):17582-17587.
[40]Kubota C, Yamakuchi H, Todoroki J, et al. Six cloned calves produced from adult fibroblast cells after long-term culture. Proc Natl Acad Sci USA,2000,97(3):990-995.
[41]Wakayama T, Tabar V, Rodriguez I, et al. Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science,2001,292(5517):740-743.
[42]Wrenzycki C, Lucas-Hahn A, Herrmann D, et al. In vitro production and nuclear transfer affect dosage compensation of the X-linked gene transcripts G6PD, PGK, and Xist in preimplantation bovine embryos. Biol Reprod,2002,66(1):127-134.
[43]吴昊.延边黄牛体细胞克隆胚胎的核型分析：[硕士学位论文].龙井：延边大学农学院,2009.
[44]Abe H, Yamashita S, Satoh T, et al. Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serumfree or serumcontaining media. Mol Reprod Dev,2002,61:57-66.
[45]敬晓棋.山羊早期胚胎端粒酶活性测定及克隆超微结构观察：[硕士学位论文].陕西咸阳：西北农林科技大学,2005.
[46]张晓红,方南洙,崔成都.延边黄牛卵母细胞于体细胞克隆胚胎内脂滴变化规律的研究.畜牧与兽医,2008,40(10)：10-13.
[47]张晓红,崔成都,方南洙,等.延边黄牛体细胞克隆早期胚胎部分超微机构的研究.畜牧与兽医,2009,41(9)：45-48.
[1]Burdon RH. Superoxide and hydrogen peroxide in relation to mammalian cell proliferation. Free Radie Biol Med,1995,18(4): 775-794.
[2]Hunt CR, Sim JE, Sullivan SJ, et al. Genomic instability and catalase gene amplification induced by chronic exposure to oxidative stress.Cancer Res,1998,58(14):3986-3992.
[3]Morel F, Cohen Tanugi Cholley L, Brandolin G et al. The O2" generating oxidase of B lymphocytes:Epstein-Barr virus-immortalized B lymphocytes as a tool for the identification of defective components of the oxidase in chronic granulomatous disease. Biochiem Biophys Acta,1993,1182(1):101-109.
[4]Forsberg L, de Faire U, Morgenstern R. Oxidative stress, human genetic variation, and disease. Arch Biochem Biophys,2001,389(1): 84-93.
[5]Gunther T, Hollriegl V, Vormann J. Perinatal development of iron and antioxidant defence system. Trace Elem Electrolytes Health Dis,1993, 7(1):47-52
[6]Mackler B, Person RE, Nguyen TD, et al. Studies of the cellular distribution of superoxide dismutases in adult and fetal rat tissues. Free Radic Res,1998,28(2):125-129.
[7]Mover H, Ar A. Antioxidant enzymatic activity in embryos and placenta of rats chronically exposed to hypoxia and hyperoxia. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol,1997,117 (2): 151-157.
[8]Finkel T. Oxygen radicals and signaling. Curr Opin Cell Biol,1998, 10(2):248-253.
[9]Finkel T, Holbrook NJ. Holbrook. Oxidants, oxidative stress and the biology of ageing. Nature,2000,408(6809):239-247.
[10]Saltman P. Oxidative Stress:A radical view. Seminars in Hematology, 1989,26:249-256.
[11]Albina JE, Cui S, Mateo RB, et al. Nitric oxide-mediated apoptosis in murine peritoneal macrophages. J Immunol,1993,150(11): 5080-5085.
[12]周彦娇.活性氧在线粒体凋亡信号传导中的调节作用.中国运动医学杂志,2005,24(6)：746-750.
[13]Keizer HG, Pinedo HM, Schaurhuis GJ, et al. Doxorubicin (adriamycin):a critical review of free radical dependent mechanisms of cytotoxicity. Pharmacol Ther,1990,47:219-231.
[14]Bickers DR, Das M, Mukhtar H, et al. Photoenhancement of lipid peroxidation associated with the generation of reactive oxygen species in hepatic microsomes of hematoporphyrin derivative-treated rats. Cancer Res,1985,45:6328-6330.
[15]Tang FJ, Ts'o PO, Lesko SA. Simultaneous in situ detection of beta-interferon mRNA and DNA in the same cell. J Histochem Cytochem,1989,37:697-701.
[16]Fujitani Y, Kasai K, Ohtani S, et al. Effect of oxygen concentration and free radicals on in vitro development of in vitro-produced bovine embryos. J Anim Sci,1997,75:483-489.
[17]Takahashi M, Keicho K, Takahashi H, et al. Effect of oxidative stress on development and DNA damage in in-vitro cultured bovine embryos by comet assay. Theriogenology,2000,54:137-145.
[18]Hashimoto S, Minami N, Takakura R, et al. Low oxygen tension during in vitro maturation is beneficial for supporting the subsequent development of bovine cumulus-oocyte complexes. Mol Reprod Dev, 2000,57:353-360.
[19]Mizushima S, Fukui Y. Fertilizability and developmental capacity of bovine oocytes cultured individually in a chemically defined maturation medium. Theriogenology,2001,55:1431-1445.
[20]de Matos DG, Furnus CC. The importance of having high glutathione (GSH) level after bovine in vitro maturation on embryo development effect of beta-mercaptoethanol, cysteine and cystine. Theriogenology, 2000,53:761-771.
[21]布拉舍.J.发育的生物化学.严绍颐,译.北京：科学出版社,1964,164-224.
[22]郑荣梁,黄中洋.自由基医学与农学基础.北京：高等教育出版社,2001,1-14,68-79,113-123.
[23]Moskovitz J. Methionine sulfoxide reductases ubiquitous enzymes involved in antioxidant defense, protein regulation, and prevention of aging-associated diseases. Biochim Biophys Acta,2005,1703(2): 213-219.
[24]Said ElMouatassim, Guerin P, Menzeo Y. Mammalian oviduct and protection against free oxygen radicals expression of genes encoding antioxidant enzymes in human and mouse. European Journal of Obsterixs,2000,89(1):1-6.
[25]Peng Y, Yang PH, Ng SS, et al. Protection of Xenopus laevis Embryos Against Alcohol-induced Delayed Gut Maturation and Growth Retardation by Peroxiredoxin 5 and catalase. Journal of Molecular Biology,2004,340(4):819-827.
[26]陈瑗,周玫.自由基医学基础与病理生理.北京：人民卫生出版社,2002：1-34.
[27]周玫,陈瑗.自由基的产生及其在某些疾病发生中的作用.中国病理生理杂志,1987,3：176.
[28]Lee CS, Koo DB, Fang NZ, et al. Potent and stage-specific action of glutathione on the development of goat early embryos in vitro. Mol Reprod Dev,2000,57:48-54.
[29]Ikegami T, Suzuki Y, Shimizu T. et al. Model mice for tissue-specific deletion of the manganese superoxide dism utase (MnSOD) gene. Biochem Biophys Res Commun,2002,296:729-736.
[30]Marklund SL, Holme E, Hellner L. Supemxide dismutase in extracellular fluids. Clin Chim Acta,1982,126:41-51.
[31]Mannick JB, Asano K, lzumi K, et al. Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell,1994,79:1137-1146.
[32]Devi M, Das N. Antiproliferative effect of polyunsaturated fatty acids and interleukin-2 on normal and abnormal human lymphocytes. Experientia,1994,50(5):489-492.
[33]Yan Oberley LW, Zhong W, et al. Manganese-containing superoxide dismutase overexpression causes phenotypic reversion in SV40-transformed human lung fibroblasts. Cancer Res,1996,56(12): 2864-2971.
[34]Storz G, Tartaglia LA, Ames BN. Transcriptional regulator of oxidative stress-inducible genes:direct activation by oxidation. Science,1990,248(4592):189-194.
[35]Yumin Hu, Daniel G, Rosen, Yan Zhou, et al. Mitochondrial MnSOD Expression in Ovarian Cancer:Role in Cell Proliferation and Response to Oxidative Stress. JBC,2005,9:1-16.
[36]Jones SP, Hoffmeyer MR, Sharp BR, et al. Role of intracellular antioxidant enzymes after in vivo myocardial ischemia and reperfusion. Am J Physiol Heart Circ Physiol,2003,284:H277-H282.
[37]Yan C, Huang A, Wu Z, et al. Increased superoxide leads to decreased flow-induced dilation in resistance arteries of Mn-SOD-deficient mice. Am J Physiol Heart Circ Physiol,2005,288:H2225-H2231.
[38]Qi X, Lewin AS, Hauswirth WW, et al. Optic neuropathy induced by reductions in mitochondrial superoxide dismutase. Invest Ophthalmol Vis Sci,2003,44:1088-1096.
[39]Qi X, Lewin AS, Sun L, et al. SOD gene transfer protects against optic neuropathy induced by deficiency of complex I. Ann Neurol, 2004,56:182-191.
[40]Strassburger M, Bloch W, Sulyok S, et al. Heterozygous deficiency of manganese superoxide dismutase results in severe lipid peroxidation and spontaneous apoptosis in murine myocardium in vivo. Free Radic Biol Med,2005,38:1458-1470.
[41]刘永彪,刘颖,赵杰.SOD对肿瘤细胞凋亡影响和抗氧化损伤的研究：Ⅰ.SOD与DDP、ADM、VP16合用对肿瘤细胞凋亡的影响.辐射研究与辐射工艺学报,2004,22(2)：114-122.
[42]刘永彪,刘颖,赵杰.SOD对肿瘤细胞凋亡影响和抗氧化损伤的研究：Ⅱ.SOD对荷瘤小鼠脾淋巴细胞的辐射防护效应.辐射研究与辐射工艺学报,2004,22(3)：165-170.
[43]黄勇,廖彪.超氧化物歧化酶在斑马鱼早期胚胎中的表达特点.陕西理工学院学报(自然科学版),2008,24(3)：91-94.
[44]Yuan HT, Bingle CD, Kelly FJ. Differential patterns of antioxidant enzyme mRNA expression in guinea pig lung and liver during development. Biochim Biophys Acta,1996,1305(3):163-171.
[45]Gomez-Anduro GA, Barillas-Mury CV, Peregrino-Urate AB, et al. The cytosolic manganese superoxide dismutase from the shrimp Litopenaeus vannamei:Molecular cloning an expression. Dev Comp Immunol,2006,30(10):893-900.
[46]高永刚.壬基酚对水生生物抗氧化酶活性的影响：[硕士学位论文].山东青岛：中国海洋大学,2006.
[47]王会文,刘建荣,静天玉,等.微量超氧化物歧化酶邻苯三酚／Vc测活法.河北大学学报(自然科学版),2000.20(1)：61-62.
[48]Schrader M, Fahimi HD. Peroxisomes and oxidative stress. Biochimica et Biophysica Acta (BBA),2006,1763(12):1755-1766.
[49]Zamocky M, Furtmuller PG, Obin C. Evolution of catalases from bacteria to humans. Antioxidants & Redox Signaling,2008,10(9): 1527-1548.
[50]张坤生,田荟琳.过氧化氢酶的功能及研究.食品科技,2007,1：8-11.
[51]Pinsent HD. Production and utilization of catalase using Saccharomyces cerevisiae. Biochem,1948,43:193-203.
[52]Summer JB, Dounce AL. Purification and characterization of a thermostable catalase from culture broth of Thermoascus aurantiacus. J Biol Chem,1937,121:417-424.
[53]Goth L. Production of catalase and glucose oxidase by Aspergillus niger using unconventional oxygenation of culture. Clin Chim Acta, 1991,196:143.
[54]Kono Y, Fridovich I. Isolation and characterization of the pseudo-catalase of Lactobacillus plantarum-A new manganese containing enzyme. J Biol Chem,1983,258(10):6015-6019.
[55]Barynin VV, Grebenko AI. T-catalase is a nonheme catalase of extremely thermophilic bacterium Thermusthermophilus. Dokl Akad Nauk SSSR,1986,286(2):461-464.
[56]赵保路.氧自由基和天然抗氧化剂.北京：科学出版社,1999.
[57]张波,刘承芸,孟紫强.二氧化硫吸入对小鼠各脏器过氧化氢酶活性的影响.环境与职业医学,2003,20(1)：10-12.
[58]冯维杨,李俞锦,钱忠义.超氧化物歧化酶和过氧化氢酶抗葡萄糖缺失诱导的神经元损伤作用研究.昆明医学学报,2009,(7)：11-15.
[59]Glenn S, Gerhard, Elizabeth J, et al. Molecular cloning and sequence analysis of theDanio reriocatalase gene. Comp Biochem Physiol B Biochem Mol Biol,2000,127:447-457.
[60]Chen X, Liang H, Remmen HV, et al. Catalase transgenic mice: characterization and sensitivity to oxidative stress. Archives of Biochemistry and Biophysics,2004,422:197-210.
[61]Gerhard GS, Kauffman EJ, Grundy MA. Molecular cloning and sequence analysis of theDanio reriocatalase gene.Comp Biochem Physiol B Biochem Mol Biol,2000,127(4):447-457.
[62]雷厚成,李毓琦,魏素萱,等.过氧化氢酶活性的化学发光法测定.华西药学杂志,1994,9(3)：176-178.
[63]徐镜波,郎佩珍.过氧化氢酶活性及活性抑制的测定.环境化学,1994,13(3)：279-282.
[64]彭志英,蒋黎.紫外速率直接法测定过氧化氢酶活性.华西医学,1995,10(1)：4-7.
[65]陈大义.催化分光光度法快速测定血清过氧化氢酶活性.四川省卫生管理干部学院学报,2000,19(4)：249-251.
[66]周强,曹春艳.血清过氧化氢酶的比色测定.哈尔滨医科大学学报。2001,35(6)：473-474.
[67]禹邦超,刘德立.应用酶学导论.武汉：华中师范大学出版社,1994,12 (1).
[68]董泗建,刘昌玲.一种鉴定过氧化氢酶活性的铁染色法.生物化学与生物物理进展,1996,23(1)：86-88.
[69]薛颖,刘平,刘泰然,等.酶-氧电极测定固定食品中H2O2方法的研究.卫生研究,2006,35(3)：313-316.
[70]姜招峰,周翔.血清过氧化氢酶活性快速测定的新方法—极谱氧电极法.白求恩医科大学学报,1989,15(5)：464-467.
[71]潘建国,王开发,郑尧隆,等.用Warburg法测定蜂花粉中过氧化氢酶活性.植物学报,2001,18(4)：505-508.
[72]罗泽娇,张随成.简易气量法测定土壤过氧化氢酶活性的研究.地质科技情报,2005,24(4)：87-90.
[73]陈瑷,周玫.生命的化学.1983；3(6)：16.
[74]蔡晓波,陆伦根.谷胱甘肽过氧化物酶与肝脏疾病.世界华人消化杂志,2009,17(32)：3279-3282.
[75]苗雨辰,白玲,苗琛,等.植物谷胱甘肽过氧化物酶研究进展.植物学通报,2005,22(3)：350-356.
[76]张舸,田东萍,苏敏,等.长期低硒、低碘对发育期大鼠肝脏和全血GSH-Px活性的影响.环境与职业医,2005,22：309-311.
[77]孙娟,陈瑗,周玫.GPx对活性氧诱发细胞程序性死亡的影响.生物化学与生物物理进展,1998,25(3)：259-262.
[78]Fukuhara R, Kageyama T. Tissue distribution, molecular cloning, and gene expression of cytosolic glutathione peroxidase in Japanese monkey. Zoolog Sci,2003,20(7):861-868.
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