凋亡抑制剂Z-VAD-FMK在猪卵母细胞冷冻保存中的应用

摘要

在猪MII期猪卵母细胞冷冻解冻后的孵育液中添加凋亡抑制剂Z-VAD-FMK,通过检测线粒体膜电位、早期凋亡、胚胎发育和相关基因表达等研究Z-VAD-FMK对冷冻后卵母细胞的影响.结果表明:(1)冷冻后线粒体膜电位(0.91)与新鲜卵母细胞(1.33)相比显著降低,添加Z-VAD-FMK显著提高了线粒体膜电位(1.19);(2)冷冻后卵母细胞的早期凋亡率(57.65％)与新鲜卵母细胞(8.37％)相比显著升高,存活率(61.45％)、孤雌激活卵裂率(13.18％)和囊胚率(3.10％)与新鲜卵母细胞(98.97％、89.41％和41.40％)相比显著下降,冻后孵育液中添加Z-VAD-FMK处理能显著降低其早期凋亡率(32.82％),并显著提高其存活、孤雌激活卵裂和囊胚的发育能力(73.21％、39.83％和10.79％);(3)冷冻后死亡受体途径和线粒体途径中促凋亡基因表达显著增加,抗凋亡基因表达显著降低,加入Z-VAD-FMK后促凋亡基因表达显著下降,抗凋亡基因表达显著增加.总之,在冻后孵育液中添加凋亡抑制剂Z-VAD-FMK能提高卵母细胞膜电位水平,降低细胞凋亡和相关基因表达,从而达到提高冻后猪卵母细胞体外发育能力的目的.%In porcine MII stage,the apoptotic inhibitor Z-VAD-FMK was added to the incubation fluid of porcine oocytes after freezing and thawing,and the effects of Z-VAD-FMK on frozen oocyte were studied by detecting the mitochondrial membrane potential (ΔΨm),early apoptotic status,embryonic development and related gene expression.The results showed:1)Mitochondrial ΔΨm in vitrified group (0.91)was significant lower than that in fresh group (1.33),Z-VAD-FMK addition after warming significantly increased mitochondrial ΔΨm (1.19).2)The early apoptotic rate in vitrified group (57.65％)was significant higher than that in fresh group (8.37 ％),and the survival rate (61.45 ％),cleavage rate (13.18 ％) and blastocyst rate (3.10％) were significant lower than those in fresh group (98.97％,89.41％ and 41.40％).Z-VAD-FMK addition significantly decreased early apoptotic rate (32.82％),and significantly increased survival rate (73.21％),cleavage rate (39.83％) and blastocyst rate (10.79％) respectively.3) Vitrification significantly increased the expression levels of pro-apoptotic related genes and decreased the expression levels of anti-apoptotic genes from both death recepotor and mitochondrial mediated apoptotic pathways.Z-VAD-FMK addition could increase anti-apoptotic gene expression and decrease pro-apoptotic gene expression.In summary,the addition of apoptotic inhibitor Z-VAD-FMK to incubation solution after vitrification could improve mitochondrial ΔΨm,reduce apoptosis and change apoptotic related gene expression levels,and then improve the in vitro developmental competence after parthenogenetic activation.