【作者】 戢爽；

【导师】 姜怀志；

【作者基本信息】 吉林农业大学 ， 动物遗传育种与繁殖， 2007， 硕士

【摘要】 VEGF基因能特异性地促进内皮细胞的增殖和血管新生。在雌性动物的的卵泡发育、卵母细胞的成熟、黄体的形成与功能的维持、早期胚胎植入、子宫蜕膜化及胎盘的形成等生理过程中，VEGF都起着十分重要的作用。因此进一步了解成年绵羊母羊在妊娠不同时期生殖器官，包括卵巢上的卵泡、输卵管及子宫内膜上VEGF基因的表达变化规律以及在高繁殖力的小尾寒羊和低繁殖力的东北细毛羊子宫内膜中VEGF基因的表达差异。探讨VEGF在绵羊母羊妊娠发育过程中的作用。初步阐明VEGF基因对绵羊妊娠发育的影响与调控机制。不仅可为反刍动物高效繁育调控与提高动物繁殖潜力提供理论指导，同时也可为开发新的调控繁殖性能的疫苗或药物奠定基础。本研究采用了反转录-聚合酶链式反应（RT-PCR）方法，分别从不同孕期的东北细毛羊和小尾寒羊的卵巢上的卵泡、输卵管和子宫内膜中提取总RNA扩增VEGF基因，通过T₄DNA连接酶将其直接连接于克隆质粒载体pMD-19-T中，转化至受体菌JM109中，通过质粒提取、PCR鉴定、序列测定，证明重组质粒pMD-19-T-VEGF中含有VEGF基因，并以β-Actin作为内源性内标检测了VEGF的mRNA在孕期绵羊卵巢上的卵泡、输卵管和子宫内膜中的表达情况，并对VEGF的部分cDNA片段进行序列测定。以期阐明VEGF基因在不同繁殖力的不同妊娠时期绵羊主要生殖器官中的作用，研究结果表明：绵羊妊娠前期、中期、后期子宫内膜中VEGFmRNA均有强烈表达，随着妊娠的持续，子宫内膜的表达逐渐升高，且妊娠早期、中期、晚期相比子宫内膜中VEGFmRNA表达差异显著（P＜0.05）。而在输卵管和卵巢上的卵泡的变化不明显。绵羊在妊娠时期子宫内膜上VEGF基因的表达水平明显高于妊娠时卵巢上的卵泡和输卵管的表达水平（P＜0.05），而输卵管的表达水平又明显高于卵巢上的卵泡的表达水平（P＜0.05）。同时，VEGFmRNA在东北细毛羊与小尾寒羊妊娠同一时期的子宫内膜、卵巢上的卵泡、输卵管相比差异不显著。测序结果显示实验羊的部分核苷酸序列与人（81bp）的同源性为96.30％，人340位的碱基为G，而绵羊相对于该位点的碱基突变为A，人363位的碱基为C，而绵羊相对于该位点的碱基突变为T，人370位的碱基为C，而绵羊相对于该位点的碱基突变为T。更多还原

【Abstract】 Vascular Endothelial Growth Factor has the role of stimulating the proliferation of epithelial cells and angiogenisis. It plays important roles in the course of female reproduction, including follicular development, oocyte maturation, corepus luteum formation and function, early embryo development; embryo implantation, uterus degeneration and placentation and so on..So making further known about the laws of Vascular Endothelial Growth Factor changes to the reproduction cycle in female ovine reproductive organs,ovary, oviduct and endometrium,and the changeble between Small Tail Han Sheep and Northeast China Fine-wool Sheep are very important.The study explained the role of VEGF during gestation and illuminated the effect of Vascular Endothelial Growth Factor in the gestation development.the research will promote the further study on genetic mechanism of development.The study on the expression of Vascular Endothelial Growth Factor in ovary, oviduct,endometrium not only bring the theory base for the high propagate and breeding regulation and reproductive protential but also establish the foundation of developing vaccine and medicine for the reproductive performance.To study the role of Vascular Endothelial Growth Factor mRNA in female reproductive organ of ovine reverse transcription-polymerase chain ruction (RT-PCR)was used to examine the expression of Vascular Endothelial Growth Factor mRNA in the ovary, oviduct and utents. The total RNA of porcine rotavirus as template, Vascular Endothelial Growth Factor gene was cloned by the reverse transcription-polymerase chain reaction (RT-PCR). The products of RT-PCR were ligated with Vector pMD-19-T, and the tigation product was used to transform E.coli JM109. By the analyses of restriction endonuclease, sequencing and PCR, it showed that the inserted gene had been a positive reading frame.We expect to clarify the role of the Vascular Endothelial Growth Factor in reproductive organs in the sheep of different reproduction and different gestion.The expression of VEGF mRNA is strongly in endometrium during the whole pregnancy, the more consistent of pregnancy, the higher level of the Vascular Endothelial Growth Factor.There is significant difference among early, midieum and late pregancy(p＜0.05).But no significant difference can be seen in the ovary and oviduct.the Vascular Endothelial Growth Factor level of endometrium is remarbly higher than that of in ovary and oviduct(p＜0.05). and the Vascular Endothelial Growth Factor level of oviduct is higher than that of in ovary(p＜0.05).The expression of the Vascular Endothelial Growth Factor Mma is no remarkble difference in the ovary ,oviduct,endometrium between Small Tail Han sheep and Northeast Fine-wool sheep during the same pregnancy. Homology of nucleofides(81 bp) of VEGF cDNA are 96.30% between sheep and human, the position of mutation is on the 340th base, from G to A.the position of mutation is on the 363th base, from C to T.the position of mutation is on the 370th base, from C to T.更多还原