血管内皮生长因子家族及其受体在猪孤雌胚胎中表达的相关研究

发布时间：2018-04-23 04:08

本文选题：孤雌发育 + 孤雌激活　；参考：《吉林大学》2014年硕士论文

【摘要】：近来，猪已经被作为研究人类疾病和干细胞治疗等的一种重要的动物模型。而猪孤雌胚胎由于有着无需受精且不违背伦理道德的优势，受到了人们的广泛关注。但是通过研究，人们发现猪的孤雌胚胎由于胎盘的缺陷而无法发育到期。目前，人们认为猪的孤雌胎儿之所以存在发育异常，一部分是由于缺少母源印记基因所导致的，而这些印记基因的表达异常则与它们的启动子甲基化水平有着关联。有报道还指出孤雌胎盘还存在着明显的血管发育缺陷，因此我们推测，孤雌胎盘的血管发育缺陷可能也是导致孤雌胎儿的发育不到期的重要因素。本实验首先通过孤雌激活技术获得了3组猪孤雌胚胎，数量分别为217，238和190个，然后通过胚胎移植将它们分别植入到3头发情的10月龄大白母猪（P-1, P-2和P-3）体内；同时，利用人工授精方法，对同期发情的3头10月龄大白猪（N-1，N-2和N-3）进行授精。在怀孕第28天（D28），通过B超发现母猪均已怀孕。然后在当天便将母猪分别麻醉，取出母猪的子宫并分离出子宫内的胎儿和胎盘。其中，母猪P-1, P-2和P-3分别的产仔数为6，8和11；母猪N-1，N-2和N-3中的胚胎数目分别为15，13和16。通过数据对比，发现母猪所怀的孤雌胎儿的数量要低于正常胎儿数量，这说明了猪孤雌胎儿的出生率可能是低于正常胎儿的。另外，通过对采集到的胎儿胎盘进行形态学对比，发现，猪孤雌胎儿和胎盘的尺寸要明显小于正常对照组，而且孤雌胎盘的颜色明显比对照组浅，结合前人的研究，我们的结果同样验证了孤雌胎盘的血管发育是有明显缺陷的。基于形态学上血管发育的不足，我们假设影响血管发育的重要因子血管内皮生长因子（VEGF）家族的表达可能与猪孤雌胎盘的发育缺陷有关联。因此，利用Q-PCR对猪孤雌胎儿胎盘中四个VEGF家族及其受体（VEGFA，PLGF，KDR和FLT-1）的表达模式做了检测，结果发现在孤雌胎儿中，这四个因子的表达均无明显变化（p0.05）；而在孤雌胎盘中，除了VEGFA的表达无显著变化之外（p0.05），KDR，FLT-1和PLGF都呈现明显的低表达（p0.05）。这说明VEGF家族可能在孤雌胎盘发育过程中起到关键作用，，进而影响孤雌胎儿的后期发育。报道证实，VEGFA的亚型通过与其受体结合，进而以不同方式激活VEGF信号通路，从而完成其对血管生成的作用。据此，我们首先利用Q-PCR对孤雌胎儿胎盘中VEGF在猪中的三个亚型VEGF120，VEGF164和VEGF188的表达做了分析，结果表明，在孤雌胎儿中，三个亚型的表达无明显变化（p0.05）；而在孤雌胎盘中，VEGF164和VEGF188呈低表达，而VEGF120的表达出现了异常升高（p0.05）。同时，利用RT-PCR对孤雌胎儿胎盘中VEGF亚型的表达进行了验证，发现与Q-PCR的结果完全相符。所以我们推测，VEGF家族及其受体在孤雌胎盘中的低表达，尤其是VEGF亚型的表达紊乱，可能影响了猪孤雌胎盘的血管生成过程，造成了孤雌胎盘发育的缺陷，进而导致了孤雌胚胎的早期发育阻滞。
[Abstract]:Recently , pigs have been used as an important animal model for the study of human diseases and stem cell therapy .

In this experiment , three groups of pigs were obtained by single - female activation technique . The numbers were 217 , 238 and 190 respectively , then they were implanted into 3 - month - old white sows ( P - 1 , P - 2 and P - 3 ) respectively by embryo transfer .
At the same time , the insemination of three 10 - month - old white pigs ( N - 1 , N - 2 and N - 3 ) in the same period was carried out by means of artificial insemination . On the 28th day of pregnancy ( D28 ) , the sows were found to have become pregnant . Then , the sows were anesthetized and the uterus of the sow was taken out and the fetus and placenta in the uterus were isolated .
It was found that the birth rate of isolated female fetus was lower than that of the normal control group . In addition , it was found that the size of the isolated female fetus was significantly lower than that of the normal control group , and the color of the isolated female placenta was significantly smaller than that of the control group .

The expression pattern of vascular endothelial growth factor ( VEGF ) , which is an important factor affecting the development of blood vessels , may be associated with the developmental defects of the isolated pig ' s placenta .
However , the VEGF family could play a key role in the development of solitary female placenta , which could affect the late development of the solitary female fetus .

The expression of VEGF , VEGF164 and VEGF188 in human fetal placenta was analyzed by Q - PCR , and the results showed that the expression of VEGF , VEGF164 and VEGF188 in the placenta of isolated female fetus was not significantly changed ( p < 0.05 ) .
The expression of VEGF164 and VEGF188 was low in isolated female placenta , while the expression of VEGF120 increased abnormally ( p < 0.05 ) . At the same time , RT - PCR was used to verify the expression of VEGF isoforms in the placenta of isolated female fetus .

Therefore , we have speculated that the low expression of VEGF family and its receptor in isolated female placenta , especially the expression of VEGF subtype , may affect the angiogenesis process of isolated female placenta , which leads to the development of solitary female placenta , which leads to the early development block of the solitary female embryo .

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：Q78

【共引文献】