【摘要】：胚胎培養(yǎng)基的成分是影響動物胚胎質量和發(fā)育命運的關鍵因素。與體內自然的發(fā)育環(huán)境相比,體外培養(yǎng)基支持胚胎發(fā)育的效率和質量還不夠理想。在培養(yǎng)基中添加生長因子可以改善卵母細胞的培養(yǎng)環(huán)境、促進胞質同步成熟,提高胚胎發(fā)育的質量。血管內皮生長因子具有促進內皮細胞增生、促進細胞遷徙和提高血管通透性等生物學作用,研究表明血管內皮生長因子與豬著床前早期胚胎體外發(fā)育有密切的相關性,但所得結果卻不相一致,這主要是因為培養(yǎng)基中含有的血清等成分未知物質對胚胎早期的生長發(fā)育具有較大的干擾。本研究首次采用了成分明確的化學限定培養(yǎng)基來研究VEGF對豬著床前早期胚胎體外發(fā)育的真實影響,再通過細胞凋亡染色技術探究VEGF對豬胚胎發(fā)育質量的影響及作用機制,從而準確的表達外源性添加生長因子對其發(fā)育的真實影響。具體實驗及結果如下:實驗一旨在通過向限定性成熟培養(yǎng)基中添加VEGF(對照組:0ng/mL;實驗組:5、25、50ng/mL),研究其對豬卵母細胞(顆粒細胞包裹≥3層)成熟及其孤雌胚胎后期發(fā)育的影響。結果表明,添加VEGF對豬卵丘細胞擴散、卵母細胞成熟、及其孤雌胚胎的卵裂率、成熟率、囊胚總細胞數、凋亡細胞數和凋亡指數均沒有顯著影響。實驗二在實驗一的基礎上,進一步向限定性成熟培養(yǎng)基中添加VEGF(對照組:0ng/mL;實驗組:5、25、50ng/mL),研究其對豬卵母細胞(顆粒細胞包裹≤1層)成熟及其孤雌胚胎后期發(fā)育的影響。結果表明,添加VEGF對豬卵丘卵母細胞細胞核成熟均無顯著影響,添加5ng/mLVEGF能夠顯著提高其成熟率,添加25ng/mLVEGF能夠顯著提高其第7天和最大囊胚率并且顯著降低其囊胚細胞凋亡指數,添加50ng/mLVEGF能夠顯著提高其第7天囊胚率。實驗三旨在通過向限定性胚胎培養(yǎng)基(PZM-4)中添加VEGF(對照組:0ng/mL;實驗組:5、25、50ng/mL),研究其對豬孤雌胚胎著床前發(fā)育及囊胚質量的影響。結果表明,添加5、25、50ng/mLVEGF均能夠顯著提高其卵裂率,添加5ng/mLVEGF能夠顯著提高其第6天、第7天和最大囊胚率,添加25ng/mLVEGF能夠顯著提高其第7天囊胚率,添加不同濃度的VEGF對豬孤雌胚胎的囊胚總細胞數、凋亡細胞數和凋亡指數均沒有顯著影響。實驗四在實驗三的基礎上,進一步研究不同時間添加5ng/mLVEGF對豬孤雌胚胎著床前發(fā)育及囊胚質量的影響。結果表明,全程添加5ng/mLVEGF能夠顯著提高其卵裂率,全程和后半程添加5ng/mLVEGF能夠顯著提高其第7天囊胚率,全程添加5ng/mLVEGF能夠顯著提高其最大囊胚率,不同時間添加5ng/mLVEGF對豬孤雌胚胎的囊胚總細胞數、凋亡細胞數和凋亡指數均沒有顯著影響。綜上所述,本研究首次在成分明確的化學限定性培養(yǎng)基中揭示了VEGF對豬著床前早期胚胎體外發(fā)育的真實影響。在促進豬卵母細胞(顆粒細胞包裹≤1層)體外成熟方面,添加5ng/mLVEGF能夠顯著提高成熟率,添加25ng/mLVEGF能夠顯著提高其囊胚率,并顯著降低細胞凋亡指數。在促進豬孤雌胚胎發(fā)育方面,添加5ng/mLVEGF能夠顯著提高囊胚率并降低凋亡細胞數,并且驗證VEGF是在胚胎發(fā)育后半程(4-7天)發(fā)揮明顯作用。總之,本研究首次在化學限定性培養(yǎng)基中驗證了VEGF對豬卵母細胞(顆粒細胞包裹≤1層)成熟和孤雌胚胎發(fā)育均有一定的促進作用,并能夠抑制其細胞凋亡,為VEGF在豬早期胚胎發(fā)育的進一步研究及提高豬體外胚胎生產效率奠定了堅實的基礎。
[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.
【學位授予單位】：安徽農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S828

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