【作者】 聂昊；

【导师】 王宗礼； 朱化彬；

【作者基本信息】 甘肃农业大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 采用现代胚胎生物工程技术是使畜牧业向高产、高效、优质、集约化发展的有效途径。但是,体外受精胚胎和克隆胚胎发育率低是制约这两项技术走向生产实践的最大障碍。本研究借助免疫荧光染色的方法,从中心体复制的关键调控基因CDK2和P53入手,研究CDK2和P53在经体外成熟18-26小时的牛成熟卵母细胞中的亚细胞定位,揭示它们在中心体复制过程中位置,分析CDK2和P53在牛成熟卵母细胞中的表达情况,从中心体异常复制的角度阐明体外受精胚胎和克隆胚胎染色体的不稳定性。研究结果如下:研究哺乳动物胚胎的免疫荧光染色条件和步骤。通过对小鼠受精卵中CDK2、P53和γ-tubulin蛋白的免疫荧光染色研究,对免疫荧光染色的条件和步骤不断优化,修改、简化和发展,摸索出一套完善的针对卵母细胞的免疫荧光染色步骤。在小鼠受精卵1细胞G1期,通过固定、封闭、染色、在荧光显微镜下观察,发现CDK2、P53和γ-tubulin蛋白均出现染色阳性。CDK2和P53在细胞有丝分裂G1期定位于中心体,分布于细胞核区。分别培养18h,22h,26h后的牛成熟卵母细胞,通过与试验一相同的固定、封闭、染色,在荧光显微镜下观察发现,(1)培养18h后的牛成熟卵母细胞,CDK2、P53和γ-tubulin蛋白均出现染色阴性。(2)培养22h后的牛成熟卵母细胞,大多数卵母细胞CDK2、P53和γ-tubulin蛋白出现染色阴性,仅有少数卵母细胞CDK2、P53和γ-tubulin蛋白出现染色阳性。(3)培养26h后的牛成熟卵母细胞,大多数卵母细胞CDK2、P53和γ-tubulin蛋白出现染色阴性,仅有少数卵母细胞CDK2、P53和γ-tubulin蛋白出现染色阳性。(4)在出现染色阳性的牛成熟卵母细胞中,CDK2和P53蛋白定位于中心体,分布于细胞核区。CDK2和P53蛋白是与细胞中心体复制密切相关的两个蛋白,其功能和细胞的正常分裂发育有关。在用于牛体外胚胎生产的牛卵母细胞中,CDK2和P53蛋白的正常表达与否,是直接影响牛体外胚胎生产效率的原因之一。更多还原

【Abstract】 Embryo adoption of modern bio-engineering technology is to enable livestock to the high-yield,high efficiency,high-quality,intensive development of an effective way.However,in vitro fertilization and embryo cloning and embryo development constraints is the low rate of these two techniques to produce the greatest obstacle to practice.This study by immune staining methods,from the Centre copy of the key regulatory genes start CDK2 and P53,and P53 on CDK2 in the 18-26 hours of in vitro mature cattle mature oocytes in the subcellular localization,and reveal them in the centre Copy of the process of location,of CDK2 and P53 in cattle mature oocytes of expression,from the centre of abnormal copy clarify the perspective of in vitro fertilization and embryo cloning and embryo chromosome instability.The results are as follows:Study of the mammalian embryo immune staining conditions and steps.Through the fertilized eggs of mice CDK2,P53 andγ-tubulin protein the immune staining on the immune staining the conditions and steps to continuously optimize,modify, simplify and development,worked out a comprehensive view of the immune oocytes Staining steps.1 cells in mouse embryos G1 phase,through a fixed,closed,dyeing,in the fluorescent microscope observation,found that CDK2,P53 andγ-tubulin protein were found positive.CDK2 and P53 in cell mitosis G1 phase of positioning at the centre,located in the nucleus area.Training were 18 h,22h,26h after the cattle mature oocytes,with the same test a fixed,closed,dyeing,in the fluorescent microscope observed that(1) 18 h after the train cattle mature oocytes,CDK2,P53 andγ-tubulin protein were found negative.(2) 22 h after the train cattle mature oocytes,the majority of oocytes CDK2,P53 andγ-tubulin protein were negative in only a small number of oocytes CDK2,P53 andγ-tubulin protein appears positive staining.(3) 26 h after the train cattle mature oocytes,the majority of oocytes CDK2,P53 andγ-tubulin protein were negative in only a small number of oocytes CDK2,P53 andγ-tubulin protein appears positive staining.(4) were positive in the event of the cattle mature oocytes,CDK2 and P53 protein location at the centre of distribution in the nuclear area.CDK2 and P53 protein is the central body and cell reproduction of the two closely related proteins,their function and a normal cell development of the split.In vitro embryos for cattle production in cattle oocytes,CDK2 and P53 protein expression of the normal or not,is a direct impact on the efficiency of cattle production in vitro embryonic one of the reasons.更多还原