【作者】 吴凯峰；

【导师】 刘东军； 旭日干；

【作者基本信息】 内蒙古大学 ， 动物学， 2004， 硕士

【摘要】 EGF与IGF-Ⅰ均为生物体内具有重要生物学作用的生长因子，卵母细胞成熟与早期胚胎发育直接或间接地受着EGF和IGF-Ⅰ体系精确的调控。本研究利用免疫荧光和激光共聚焦扫描技术探讨了表皮生长因子受体(EGFR)，类胰岛素生长因子(IGF-Ⅰ)及其受体在牛早期胚胎中的蛋白表达。同时，在牛卵母细胞成熟培养液中添加不同量的EGF、IGF-Ⅰ，对成熟后的卵母细胞检测核成熟率，观察生长因子对卵母细胞成熟的影响以及成熟液中生长因子的最佳添加浓度，结果如下： ① 在牛早期胚胎既受精卵到囊胚的发育阶段虽然不表达EGF的蛋白，但是EGFR的蛋白一直可以检测得到。实验发现从二细胞到囊胚均可见EGFR的表达，其主要分布在细胞膜上，随着发育天数的增加，表达量在增加。文献显示在牛体内早期胚胎发育阶段的子宫和输卵管中有EGF的表达分泌，说明在早期胚胎的发育过程中，EGF对牛早期胚胎的调控采用旁分泌的形式，同时EGFR的表达在时序上具有连贯性。 ② 从二细胞到囊胚对IGF-Ⅰ、IGF-Ⅰ R的免疫结果显示，IGF-Ⅰ体系在早期胚胎发育过程中一直有表达，同样也随着发育天数的增加，表达量也在增加。IGF-Ⅰ在细胞膜与胞质内均可见，受体主要位于质膜上。结果表明IGF-Ⅰ内蒙古大学硕士学位论文中文摘要体系在牛早期胚胎发育中有生物学作用，采用自分泌方式参与调控，表达具有时序连贯性。③在卵母细胞成熟培养液中分别添加ing/ml、long/ml、song/ml、IO0ng/ml、 Zoong/ml的EGF，1 Ong/ml、50ng/ml、loong/ml、15ong/ml、Zoong/ml的 IGF一I，观察体外成熟培养22小时后的卵母细胞成熟率。结果添加有EGF 实验组的成熟率分别是36.73%、42.18%、56.18%、53.67%、56.5%，同对 照组相t匕都较对照组的成熟率(34.040，0)高，且song/ml、loong/ml、 200ng/ml实验组的成熟率同对照组差异显著(P<0.05)。IGF一I实验组的成 熟率分别是38.46%、46.94%、58.73%、55%、58.7%，50ng/ml、100ng/ml、 15ong/ml、20ong/ml都较对照组的成熟率要高(38.60%)，loong/ml、 15ong/ml、Zoong/ml实验组同对照组相比差异显著(p<0.05)。将50ng/ml的 EGF、1 00ng/ml的IGF一I共同添加在成熟培养液中得到的成熟率为75%， 同对照组相比(36.54%)差异极显著(P<0.01)。结果显示在牛卵母细胞成 熟培养液中添加EGF、IGF一I会提高卵母细胞的成熟率，并且随着浓度的增加，这种生物学作用将出现饱和性，最佳作用浓度为50ng/mlEGF、IO0ng/mllGF一I。更多还原

【Abstract】 EGF (epidermal growth factor) and IGF- I (insulin-like growth factor-I) are important growth factors to the development of preimplantation embryo ,muturation of oocyte and development of preimplantion embryo are regulated by EGF and IGF- I systems in direct or indirect manner. The aim of this study was to illuminate the expression of proteins of the EGFR, IGF- I and IGF- I R in preimplantation bovine embryos by using indirect immunofluorescence and laser scanning confocal microscopy. Furthermore,Bovine follicular oocytes were matured in vitro and for 22 h in TCM-199 (tissue culture medium-199) without or with EGF (1,10,50 ng/ml), IGF-I (10,50,100 ng/ml). At the end of the culture period,the nuclear muturation rates were observed. This study was carried out to identify the possible effects of EGF and IGF-I to nuclear maturation in bovine oocytes matured in vitro and optimistic concentration that added in muturation medium.(1) There is not existing EGF protein in bovine preimplantation embryo ,but EGFR protein could be detected in this period. It was found EGFR protein existed in every stage(from two cell to blastula )and located on cell membrane. The protein expression intensity increasd with embryo development. There are experiments demonstrated EGF protein exists in uterus and oviduct during the bovine preimplantation embryo developmental period, it indicated that EGFcould effect preimplantation embryo development by paracrine manner. (2)Immunofluorescence result demonstrate that IGF- I system have expressed in preimplantation embryo deveopmental period. The proteins expression intensity also increasd with embryo development. IGF- I can be detected in the cell membrance and cell plasma, IGF- I R was located in cell membrance. The results indicated that IGF- I system have biological action to bovine embryo development. (3)EGF concentrated in Ing/ml, 10ng/ml, 50ng/ml,100ng/ml,200ng/ml and IGF-I concentrated in 10ng/ml, 50ng/ml, 100ng/ml,150ng/ml,200ng/ml were added in muturation medinm, then the nuclear muturation rates after cultured 22 hours were evaluated. The rates of nuclear maturation supplemented with EGF were 36.73%,42.18%,56.18%,53.67%,56.5% respectively,were all higher than the rate of contrast(34.04%).The rates of nuclear maturation supplemented with IGF- I were 38.46%,46.94%,58.73%,55%,58.7% respectively, 50ng/ml,100ng/ml,150ng/ml,200ng/ml IGF- I werer higher than contrast(38.60%). The muturation rate was 75% when 50ng/ml EGF plus 100ng/ml IGF- I were added in muturation medium, and significantly higher than that of constrast(36.54%, P<0.01). The result demonstrated EGF, IGF-I could increase the bovine oocyte nuclear muturation rate and the effect were saturable (concentration=50ng/mlEGF,100ng/mlIGF- I ) .更多还原