血管内皮生长因子对猪着床前早期胚胎体外发育的影响

发布时间：2018-08-11 15:49

【摘要】：胚胎培养基的成分是影响动物胚胎质量和发育命运的关键因素。与体内自然的发育环境相比,体外培养基支持胚胎发育的效率和质量还不够理想。在培养基中添加生长因子可以改善卵母细胞的培养环境、促进胞质同步成熟,提高胚胎发育的质量。血管内皮生长因子具有促进内皮细胞增生、促进细胞迁徙和提高血管通透性等生物学作用,研究表明血管内皮生长因子与猪着床前早期胚胎体外发育有密切的相关性,但所得结果却不相一致,这主要是因为培养基中含有的血清等成分未知物质对胚胎早期的生长发育具有较大的干扰。本研究首次采用了成分明确的化学限定培养基来研究VEGF对猪着床前早期胚胎体外发育的真实影响,再通过细胞凋亡染色技术探究VEGF对猪胚胎发育质量的影响及作用机制,从而准确的表达外源性添加生长因子对其发育的真实影响。具体实验及结果如下:实验一旨在通过向限定性成熟培养基中添加VEGF(对照组:0ng/mL;实验组:5、25、50ng/mL),研究其对猪卵母细胞(颗粒细胞包裹≥3层)成熟及其孤雌胚胎后期发育的影响。结果表明,添加VEGF对猪卵丘细胞扩散、卵母细胞成熟、及其孤雌胚胎的卵裂率、成熟率、囊胚总细胞数、凋亡细胞数和凋亡指数均没有显著影响。实验二在实验一的基础上,进一步向限定性成熟培养基中添加VEGF(对照组:0ng/mL;实验组:5、25、50ng/mL),研究其对猪卵母细胞(颗粒细胞包裹≤1层)成熟及其孤雌胚胎后期发育的影响。结果表明,添加VEGF对猪卵丘卵母细胞细胞核成熟均无显著影响,添加5ng/mLVEGF能够显著提高其成熟率,添加25ng/mLVEGF能够显著提高其第7天和最大囊胚率并且显著降低其囊胚细胞凋亡指数,添加50ng/mLVEGF能够显著提高其第7天囊胚率。实验三旨在通过向限定性胚胎培养基(PZM-4)中添加VEGF(对照组:0ng/mL;实验组:5、25、50ng/mL),研究其对猪孤雌胚胎着床前发育及囊胚质量的影响。结果表明,添加5、25、50ng/mLVEGF均能够显著提高其卵裂率,添加5ng/mLVEGF能够显著提高其第6天、第7天和最大囊胚率,添加25ng/mLVEGF能够显著提高其第7天囊胚率,添加不同浓度的VEGF对猪孤雌胚胎的囊胚总细胞数、凋亡细胞数和凋亡指数均没有显著影响。实验四在实验三的基础上,进一步研究不同时间添加5ng/mLVEGF对猪孤雌胚胎着床前发育及囊胚质量的影响。结果表明,全程添加5ng/mLVEGF能够显著提高其卵裂率,全程和后半程添加5ng/mLVEGF能够显著提高其第7天囊胚率,全程添加5ng/mLVEGF能够显著提高其最大囊胚率,不同时间添加5ng/mLVEGF对猪孤雌胚胎的囊胚总细胞数、凋亡细胞数和凋亡指数均没有显著影响。综上所述,本研究首次在成分明确的化学限定性培养基中揭示了VEGF对猪着床前早期胚胎体外发育的真实影响。在促进猪卵母细胞(颗粒细胞包裹≤1层)体外成熟方面,添加5ng/mLVEGF能够显著提高成熟率,添加25ng/mLVEGF能够显著提高其囊胚率,并显著降低细胞凋亡指数。在促进猪孤雌胚胎发育方面,添加5ng/mLVEGF能够显著提高囊胚率并降低凋亡细胞数,并且验证VEGF是在胚胎发育后半程(4-7天)发挥明显作用。总之,本研究首次在化学限定性培养基中验证了VEGF对猪卵母细胞(颗粒细胞包裹≤1层)成熟和孤雌胚胎发育均有一定的促进作用,并能够抑制其细胞凋亡,为VEGF在猪早期胚胎发育的进一步研究及提高猪体外胚胎生产效率奠定了坚实的基础。
[Abstract]:The composition of embryo culture medium is the key factor affecting the quality and fate of animal embryos.Compared with the natural development environment in vivo,the efficiency and quality of in vitro culture medium supporting embryo development are not satisfactory enough.Adding growth factor into culture medium can improve the culture environment of oocytes,promote the synchronous maturation of cytoplasm and improve embryo development. Vascular endothelial growth factor (VEGF) has the biological functions of promoting endothelial cell proliferation, promoting cell migration and enhancing vascular permeability. Studies have shown that vascular endothelial growth factor is closely related to the in vitro development of preimplantation embryos in pigs, but the results are not consistent, mainly because of the blood contained in the medium. The unknown substances in clear composition have great interference on the early growth and development of embryos. For the first time in this study, a chemically defined medium with a well-defined component was used to study the real effects of VEGF on the early development of preimplantation embryos in pigs, and then the effects of VEGF on the quality of pig embryo development and the mechanism of action were explored through apoptosis staining technology. The experimental results were as follows: Experiment 1 was designed to investigate the maturation of porcine oocytes (granulosa cells wrapped in more than 3 layers) and the development of their parthenogenetic embryos by adding VEGF to the defined mature medium (control group: 0ng/mL; experimental group: 5,25,50ng/mL). The results showed that the addition of VEGF had no significant effect on the proliferation of porcine cumulus cells, oocyte maturation, the cleavage rate of the parthenogenetic embryos, the number of blastocyst cells, the number of apoptotic cells and the apoptotic index. Experiment two, on the basis of Experiment 1, further added VEGF to the restricted mature medium (control group: 0ng/mL; experimental group: 5). 25,50ng/mL) to study the effects of the addition of VEGF on the maturation of porcine oocytes (granulosa cells wrapped in less than 1 layers) and the development of parthenogenetic embryos. The results showed that the maturation rate of the cumulus oocytes was not significantly affected by addition of 5ng/mLVEGF, and the maturation rate was significantly increased by adding 5ng/mLVEGF. The addition of 25ng/mLVEGF could significantly increase the maximum number of eggs and the largest sac. The rate of blastocyst apoptosis was significantly decreased and the blastocyst apoptosis rate was significantly increased by adding 50ng/mLVEGF. The aim of experiment three was to add VEGF to the restrictive embryo culture (PZM-4) (control group: 0ng/mL; experimental group: 5,25,50ng/mL) to study the effect of the addition of VEGF on the development of preimplantation embryos and the quality of blastocysts. The addition of 5,25,50ng/mLVEGF could significantly increase the cleavage rate. Adding 5ng/mLVEGF could significantly increase the sixth day, seventh day and maximum blastocyst rate. Adding 25ng/mLVEGF could significantly increase the blastocyst rate of seventh days. Adding different concentrations of VEGF had no significant effect on the total number of blastocysts, the number of apoptotic cells and the apoptotic index of parthenogenetic embryos. Experiment four, on the basis of experiment three, further studied the effect of adding 5ng/mLVEGF at different times on the preimplantation development and blastocyst quality of porcine parthenogenetic embryos. The results showed that adding 5ng/mLVEGF to the whole course significantly increased the cleavage rate. The addition of 5ng/mLVEGF during the whole and later stages could significantly increase the blastocyst rate of seventh days, and 5ng/mLVEGF could be added throughout the whole process. Significantly increased the maximum blastocyst rate. 5ng/mLVEGF at different times had no significant effect on the total number of blastocysts, apoptotic cells and apoptotic index of porcine parthenogenetic embryos. In summary, this study was the first to reveal the true effect of VEGF on the development of preimplantation early embryos of pigs in a well-defined chemical restricted medium. To promote porcine oocytes (granulosa cells wrapped in less than 1 layers) in vitro maturation, adding 5ng/mLVEGF can significantly improve the maturation rate, adding 25ng/mLVEGF can significantly improve the blastocyst rate and significantly reduce the apoptotic index. In promoting the development of porcine parthenogenetic embryos, adding 5ng/ mLVEGF can significantly improve the blastocyst rate and reduce the number of apoptotic cells. Moreover, VEGF was proved to play a significant role in the development of the embryo after half a day (4-7 days). In conclusion, this study verified for the first time in the chemical restricted medium that VEGF could promote the maturation of porcine oocytes (granulosa cells wrapped in less than 1 layers) and the development of parthenogenetic embryos, and could inhibit the apoptosis of them, which is the development of VEGF in the early stage of porcine embryo development. The study laid a solid foundation for further research on the efficiency of pig embryo in vitro.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828

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