体外成熟牛卵母细胞BMP15和GDF9的表达及其对卵丘细胞凋亡的影响

发布时间：2018-02-28 11:38

本文关键词： 牛体外成熟 BMP15 GDF9 卵丘细胞细胞凋亡　出处：《中国农业科学院》2015年博士论文　论文类型：学位论文

【摘要】：体外成熟(IVM)、体外受精(IVF)和胚胎培养(IVF)等技术是体外生产家畜、实验动物甚至野生动物胚胎的有效技术方法,是提高家畜繁殖性能的重要技术途径,因此,全世界许多研究人员开展了多种动物体外胚胎技术研究,体外胚胎生产效率不断提高。卵母细胞IVM时卵母细胞质量与发育能力是决定体外生产胚胎(IVP)效率的重要因素。体外实验表明,卵母细胞发育与其周围包裹得颗粒细胞之间相互影响,不同动物如小鼠、羊和猪的颗粒细胞(CCs)都对其卵母细胞发育能力具有重要意义,而卵母细胞分泌因子如生长分化因子9(GDF9)和骨形态发生蛋白15(BMP15)与CCs细胞凋亡息息相关,进而影响卵母细胞自身质量和受精后胚胎的发育。IVM时培养液中添加一定浓度的外源性BMP15和GDF9,CCs中细胞凋亡显著下降,因此,GDF9和BMP15可能是卵母细胞体外成熟过程中调节CCs细胞凋亡的重要因素。然而,不同动物中BMP15和GDF9调节CCs凋亡的机理可能不尽相同。因此,本论文研究牛体外成熟卵母细胞分泌因子BMP15和GDF9的表达,添加外源BMP15和GDF9对CCs相关促凋亡和抗凋亡基因表达的影响,以及外源GDF9调控CCs凋亡的可能信号通路。本论文包括三个实验:(1)体外培养牛卵母细胞BMP15和GDF9基因表达的研究。分别研究不同培养时间0h(GV)、16h(MII)、24h(MII)BMP15和GDF9基因m RNA的表达;(2)体外添加外源BMP15和GDF9对体外培养牛卵母细胞颗粒细胞CCs凋亡影响的研究。培养液中分别添加BMP15和GDF9的浓度为0BMP15+GDF9(对照组);100ng/m L BMP15;100ng/m L GDF9;100ng/m L BMP15+100ng/m L GDF9。培养24h后,定量RT-PCR分析CCs细胞中促凋亡基因(Bim、Bax、p53),和抗凋亡基因(Bcl2)m RNA的表达,Western Blot检测Bim和p53蛋白的表达;(3)GDF9调控CCs凋亡通路的研究。培养液中添加100ng/ml的GDF9,分别添加抑制剂SB203580、PDTC、SB431542或者LY294002作用1h后,Western Blot检测Bim蛋白表达。实验结果表明:(1)IVM期间BMP15和GDF9 m RNA在牛卵母细胞中表达,并在16h(MII)达到最大;(2)外源性GDF9能显著降低Bim m RNA和蛋白在牛卵丘细胞中的表达(3)GDF9可能通过SMAD、PI3K途径降低Bim的表达。
[Abstract]:In vitro maturation (IVM), in vitro fertilization (IVF) and embryo culture (IVF) technology is a effective technique of in vitro production of livestock, animal and even wild animal embryos, is an important approach to improve the reproductive performance of livestock, so many researchers around the world to carry out a variety of animal embryos in vitro embryo production in vitro continuously improve the efficiency of oocytes. IVM oocyte quality and developmental ability is determined in vitro embryo production (IVP) is an important factor of efficiency. In vitro experiments showed that oocytes and granulosa cells in vitro wrapped around to each other between different animal, such as mice, sheep and pig granulosa cells (CCs) has important significance the development ability of the oocytes and oocyte secreted factors such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) and CCs cell apoptosis closely, thereby affecting the oocytes The cell quality itself and after fertilization of the embryo development of.IVM culture medium added with a certain concentration of exogenous BMP15 and GDF9, apoptosis of CCs cells decreased significantly, therefore, GDF9 and BMP15 may be an important factor in regulating the apoptosis of CCs cells during oocyte maturation in vitro. However, the mechanism of BMP15 and GDF9 in regulating the apoptosis of CCs in different the animal may not be the same. Therefore, the expression of bovine oocytes matured in vitro secreted BMP15 and GDF9, the effects of exogenous BMP15 and GDF9 on the expression of CCs related apoptosis and anti apoptosis gene, and exogenous GDF9 regulation CCs apoptosis signal pathway. This paper consists of three experiments: (1 study on the expression of bovine oocytes) BMP15 and GDF9 gene in vitro. Studies of different culture time 0h (GV), 16h (MII), 24h (MII) expression of BMP15 and GDF9 gene m RNA; (2) in vitro with exogenous BMP15 and GDF9 on in vitro Study on the apoptosis of cattle oocyte granulosa cells CCs. BMP15 and GDF9 concentration were added to the solution of 0BMP15+GDF9 (control group); 100ng/m L BMP15; 100ng/m L GDF9; 100ng/m L BMP15+100ng/m L GDF9. 24h after culture, quantitative analysis of RT-PCR in CCs cells apoptosis genes (Bim, Bax, p53). And the anti apoptotic gene (Bcl2) expression of M RNA, the expression of Western Blot detection of Bim and p53 protein; (3) the regulatory role of GDF9 on CCs apoptosis pathway. GDF9 100ng/ml added in liquid culture, adding inhibitor SB203580, PDTC, SB431542 or LY294002 1H, to detect the expression of Bim protein Western Blot. The experimental results show that: (1) the expression of IVM BMP15 and GDF9 m during RNA in bovine oocytes, and in 16h (MII) reached the maximum; (2) exogenous GDF9 can significantly reduce the Bim m and RNA protein in bovine cumulus cells the expression of GDF9 (3) by SMAD, PI3K pathway decreased Bim table Da.

【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S823

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