旁分泌因子促进绵羊卵母细胞体外成熟和早期胚胎发育的作用研究

发布时间：2018-03-28 19:09

  本文选题：结缔组织生长因子　切入点：基质细胞衍生因子-1　出处：《内蒙古大学》2017年硕士论文

【摘要】：绵羊是非常重要的经济型家畜,为人们提供肉、奶、皮毛等产品。运用卵母细胞体外成熟(In Vitro Maturation,IVM)、体外受精(In Vito Fertilization,IVF)和胚胎移植(Embryo Transfer,ET)技术对优质家畜进行良种扩繁,能有效提高家畜生产力。卵母细胞的发育与成熟是这些技术的基础,因此,卵母细胞成熟的品质好坏直接影响子代能否成活及存活后代的健康状况。从卵母细胞成熟到早期胚胎发育的过程中,除内源性基因的表达调控作用之外,培养环境中的信号分子作用也影响其能否正常发育。通过旁分泌因子发挥调控作用是这种调控最常见、最直接的方式之一。结缔组织生长因子(Connective tissue growth factor,CTGF)、基质细胞衍生因子-1(Stromal cell-derived factor-1,SDF-1)、神经生长因子(Nerve growth factor,NGF)、肝细胞生长因子(Hepatocyte growth factor,HGF)是卵巢旁分泌因子。研究发现CTGF能促进小鼠、大鼠卵泡发育,SDF-1能增加颗粒细胞存活和提高胚胎质量,NGF能以剂量依赖的方式增强山羊初级卵泡的体外生长,HGF能诱导牛的颗粒细胞增殖。但这四种旁分泌因子是否能够作用于绵羊卵母细胞,并提高卵母细胞体外成熟率、受精率和早期胚胎发育率,尚未做过充分研究。因此,我们利用绵羊卵母细胞作为实验材料,研究CTGF、SDF-1、NGF和HGF单独添加和组合添加对绵羊卵母细胞成熟和早期胚胎发育的作用。我们在绵羊卵母细胞成熟液中分别单独添加不同浓度梯度的CTGF、SDF-1、NGF和HGF因子,统计绵羊卵母细胞成熟率,找到单独添加CTGF、SDF-1、NGF和HGF因子的最佳浓度。接着将这四种因子组合添加到绵羊卵母细胞成熟液和早期胚胎发育液中,统计绵羊卵母细胞成熟率、卵子激活率和早期胚胎发育率。以卵丘扩散指数(cumulus expansion index,CEI)、细胞膜表面早期细胞凋亡信号和胚胎的囊胚细胞数作为评价卵母细胞成熟和早期胚胎发育潜能的指标,并且检测了凋亡基因Bax、Bcl-2、P53和PTGS2的基因表达量。实验结果发现:(1)在四种旁分泌因子单独使用的实验中,添加30.0 ng/mL CTGF 因子,10.0ng/mLSDF-1 因子,3.0ng/mLNGF 因子,100.0ng/mLHGF因子能促进绵羊卵母细胞成熟和早期胚胎发育;(2)在四种旁分泌因子组合使用的实验中,添加30.0 ng/mLCTGF + 10.0 ng/mL SDF-1+3.0 ng/mLNGF+100.0ng/mL.HGF 时,卵母细胞成熟率、激活率以及早期胚胎各个时期的发育率都优于单独添加单一因子组和不添加因子组;(3)组合因子对CEI、抑制卵子凋亡率和提高囊胚细胞数有显著作用;(4)组合因子的使用能降低卵丘细胞、卵母细胞和囊胚细胞中凋亡基因的表达量。实验结果表明,旁分泌因子CTGF、SDF-1、NGF和HGF能促进卵母细胞成熟;四种因子组合使用能促进绵羊卵母细胞成熟和早期胚胎的发育。
[Abstract]:Sheep are very important economic livestock, providing people with meat, milk, fur and other products. Using in Vitro maturation in vitro maturation of oocytes, in Vito fertilization IVF) and embryo transfer transfer et) technology to improve the breeding of high quality domestic animals. The development and maturation of oocytes are the basis of these techniques. The quality of oocyte maturation directly affects the survival of offspring and the health status of surviving offspring. During the process from oocyte maturation to early embryonic development, the expression and regulation of endogenous genes are involved in the process of oocyte maturation. Signaling molecules in the culture environment also affect their normal development. Regulation through paracrine factors is the most common regulation. One of the most direct ways is that connective tissue growth factor-CTGFs, stromal cell-derived factor-1, nerve growth factor (NGF) and hepatocyte growth factor (HGFs) are the paracrine factors of ovary. SDF-1 can increase granulosa cell survival and improve embryo quality. NGF can enhance the growth of goat primary follicles in vitro in a dose-dependent manner. HGF can induce the proliferation of bovine granulosa cells. But whether these four paracrine factors can promote the proliferation of bovine granulosa cells? Can act on ovine oocytes, The rate of oocyte maturation in vitro, fertilization rate and early embryo development rate have not been fully studied. Therefore, we use sheep oocytes as experimental materials. To study the effects of HGF and CTGFF-1NGF on ovine oocyte maturation and early embryo development, we added CTGF- SDF-1NGF and HGF factor in ovine oocyte maturation fluid separately. The maturation rate of ovine oocytes was calculated, and the optimum concentration of HGF factor and SDF-1NGF were found. Then the four factors were added to ovine oocyte maturation fluid and early embryonic development fluid to calculate the ovine oocyte maturation rate. Oocyte activation rate and early embryo development rate. The cumulus expansion index CEI, early cell apoptosis signal on cell membrane surface and blastocyst number of embryos were used as indicators to evaluate oocyte maturation and early embryo development potential. The expression of apoptotic gene Bax-Bcl-2P53 and PTGS2 was detected. The results showed that the expression of Bax-Bcl-2P53 and PTGS2 was detected in four paracrine factors. Adding 30.0 ng/mL / mLSDF-1 factor and 3.0 ng / mLNGF factor 100.0ng / mLHGF can promote ovine oocyte maturation and early embryonic development. In the experiment of four paracrine factors, the oocyte maturation rate was increased by adding 30.0 ng/mLCTGF 10.0 ng/mL SDF-1 3.0 ng/mLNGF 100.0ng/mL.HGF. The activation rate and the development rate of each stage of early embryo were better than those of single factor group and no factor group. The combination factor had significant effect on CEI, inhibiting egg apoptosis rate and increasing blastocyst cell number. Use can reduce the number of cumulus cells, The expression of apoptotic genes in oocytes and blastocysts. The results showed that the paracrine factor CTGFF- SDF-1NGF and HGF could promote oocyte maturation, and the combination of four factors could promote ovine oocyte maturation and early embryo development.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S826
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本文编号：1677659