【作者】 陈雪；

【导师】 逄大欣；

【作者基本信息】 吉林大学 ， 生物工程（专业学位）， 2020， 硕士

【摘要】 目前猪的卵母细胞在体外成熟的效率较低、质量较差,导致后期胚胎体外生产的发育效率低下,严重影响了克隆技术在生物医学模型和动物繁殖等领域中的应用。卵母细胞体外成熟是体细胞核移植、体外受精等技术的主要环节,制约着后期胚胎发育的生产效率,对胚胎发育的影响至关重要。本实验主要以猪卵母细胞和孤雌激活(PA)、体细胞核移植(SCNT)、体外受精(IVF)囊胚为研究对象,旨在探究体外培养体系中,维生素A(VtiaminA,VA)对卵母细胞体外成熟及其胚胎早期发育的影响。首先,本实验对猪卵母细胞培养基中所添加的VA浓度进行了优化,发现20μg/ml的VA能显著提高猪卵母细胞的体外成熟率(75%±2.13%vs 52%±3.26%)(***P<0.01,差异极显著)。除此之外,还发现VA添加组中MⅡ期卵母细胞内RAR基因和GSH基因的转录水平均显著高于对照组。成熟后卵母细胞的免疫荧光染色结果进一步证实,VA添加组MⅡ期细胞内的ROS和GSH的抗氧化水平显著高于对照组。基于上述结果,本实验接着用VA对PA、SCNT、IVF早期胚胎的卵裂率、囊胚率的影响进行了深入研究。结果发现,与对照组相比,孤雌胚胎VA添加组的 2 细胞卵裂率(89.33%±2.89%vs 81.33%±2.53%),4 细胞卵裂率(81%±3.04%vs 70.33%±2.2%)、8 细胞卵裂(50%±2.13%vs 41.67%±2.18%)及胚胎的囊胚率(30%±3.16%vs 23.67%±2.29%)明显高于对照组(*P<0.05,差异显著);SCNT胚胎VA添加组的2细胞卵裂率(60.33%±2.34%vs 50.67%±3.23%),4细胞卵裂率(38.67%±3.54%vs 29.67%±2.27%)、8 细胞卵裂率(35.67%±2.73%vs 27.67%±3.18%)及胚胎的囊胚率(17.33%±3.29%vs 9.67%±3.16%)也明显高于对照组(*P<0.05,差异显著),以及IVF胚胎VA添加组的2细胞卵裂率(79.67%±2.34%vs 58.67%±3.23%),4 细胞卵裂率(59%±3.14%vs 49.67%±2.47%)、8 细胞卵裂率(39.33%±2.33%vs 29.67%±3.58%)及胚胎的囊胚率(21%±2.49%vs 17%±2.66%)均显著高于对照组(*P<0.05,差异显著)。最后,本实验探究了 VA对PA、SCNT、IVF囊胚的多能性、囊胚数及凋亡的影响。免疫荧光染色分析结果发现,相比较与对照组,VA添加组囊胚中,多能性基因Oct4、Cdx2、Sox2等的表达量明显上升,囊胚细胞数也有显著提高,且VA添加组的囊胚凋亡情况显著低于对照组。综上所述,本实验优化了猪卵母细胞和胚胎在体外培养体系中VA的培养浓度,并证明了优化后的VA能显著提高猪卵母细胞的体外成熟率,同时能显著提高胚胎的卵裂率、囊胚率。除此之外,发现VA添加组能增强并提高猪囊胚的细胞数和多能性,总体提高了猪卵母细胞体外成熟和胚胎发育效率。本研究有利于克隆技术在生物医学模型和动物繁殖等领域中更好的应用。更多还原

【Abstract】 At present,the efficiency of pig oocyte maturation in vitro is low and the quality is poor,which leads to the low development efficiency of late embryo production in vitro,which seriously affects the application of cloning technology in biomedical models and animal reproduction.Oocyte maturation in vitro is the main link of somatic cell nuclear transfer,in vitro fertilization and other technologies,which restricts the production efficiency of late embryonic development and is critical to the impact of embryonic development.This experiment mainly takes pig oocytes and parthenogenetic activation(PA),somatic cell nuclear transfer(SCNT),in vitro fertilization(IVF)blastocysts as the research objects,and aims to explore the role of vitamin A(VtiaminA,VA)in the in vitro culture system.Oocyte maturation in vitro and its influence on early embryonic developmentFirst,this experiment optimized the concentration of VA added to the porcine oocyte culture medium,and found that 20μg/ml VA can significantly improve the in vitro maturation rate of porcine oocytes(75%±2.13%vs 52%±3.26%)(***P<0.01,the difference is extremely significant).In addition,it was also found that the transcription level of RAR gene and GSH gene in M II stage oocytes in the VA-added group was significantly higher than that in the control group.The results of immunofluorescence staining of oocytes after maturation further confirmed that the antioxidant levels of ROS and GSH in MⅡ phase cells in the VA added group were significantly higher than those in the control groupBased on the above results,this experiment was followed by an in-depth study on the effects of VA on the cleavage rate and blastocyst rate of PA,SCNT,and IVF early embryos.It was found that compared with the control group,the 2-cell cleavage rate(89.33%±2.89%vs 81.33%±2.53%)and 4-cell cleavage rate(81%±3.04%vs 70.33%±2.2%),8-cell cleavage(50%±2.13%vs 41.67%±2.18%)and embryo blastocyst rate(30%±3.16%vs 23.67%±2.29%)were significantly higher than the control group(*P<0.05,Significant difference);2-cell cleavage rate(60.33%±2.34%vs 50.67%±3.23%),4-cell cleavage rate(38.67%±3.54%vs 29.67%±2.27%),8 Cell cleavage rate(35.67%±2.73%vs 27.67%±3.18%)and embryo blastocyst rate(17.33%±3.29%vs 9.67%±3.16%)were also significantly higher than the control group(*P<0.05,the difference was significant),and the 2-cell cleavage rate(79.67%±2.34%vs 58.67%±3.23%),4-cell cleavage rate(59%±3.14%vs 49.67%±2.47%),8-cell egg The splitting rate(39.33%±2.33%vs 29.67%±3.58%)and the embryo blastocyst rate(21%±2.49%vs 17%±2.66%)were significantly higher than those in the control group(*P<0.05,significant difference)Finally,this experiment explored the effects of VA on the pluripotency,blastocyst number,and apoptosis of PA,SCNT,and IVF blastocysts.Immunofluorescence staining analysis found that compared with the control group,the expression levels of the pluripotency genes Oct4、Cdx2、Sox2,etc.in the VA added group blastocysts increased significantly,the number of blastocyst cells also increased significantly,and the VA added group The embryo apoptosis was significantly higher than the control groupIn summary,this experiment optimized the concentration of VA in the in vitro culture system of porcine oocytes and embryos,and proved that the optimized VA can significantly increase the in vitro maturation rate of porcine oocytes,and can also significantly increase Embryo cleavage rate and blastocyst rate.In addition,it was found that the VA addition group can enhance and increase the cell number and pluripotency of porcine blastocysts,and generally improve the efficiency of porcine oocyte maturation and embryo development in vitro.This study is conducive to better application of cloning technology in the fields of biomedical models and animal reproduction.更多还原