E-64对绵羊卵母细胞体外成熟过程中caspase表达规律的影响

Effects of E-64 on caspase expression during sheep oocytes in vitro maturation

为了研究E-64对绵羊卵母细胞体外成熟过程中caspase表达的影响,从而探究E-64抑制卵母细胞凋亡的基本途径。本试验利用双夹心ELISA方法,研究体外成熟过程中绵羊卵母细胞内caspase-3,caspase-8以及caspase-9的蛋白表达水平及酶活水平的变化规律,以及成熟液中添加E-64对其产生的影响。结果表明:caspase-3、caspase-8和caspase-9的蛋白表达水平均呈先升高后降低的变化规律,caspase-3和caspase-9的活性与其蛋白表达的变化规律相一致,而caspase-8的活性则呈逐渐升高的变化规律;成熟液中添加E-64,对caspase-3、caspase-8和caspase-9的蛋白表达水平均无显著性(P>0.05)影响,但可以降低caspase-8和caspase-9的酶活性(P<0.05),而对caspase-3的酶活性无显著性影响(P>0.05)。表明E-64通过在酶活性水平上下调死亡受体途径中caspase-8的活性和线粒体途径中caspase-9的活性,从而降低了卵母细胞体外成熟过程中的细胞凋亡刺激。

This experiment was conducted to study the effects of E-64 on caspase expression during oocytes IVM,and to investigate the basic pathway that E-64 inhibits oocytes apoptosis.Double antibody sandwich ELISA was undertaken to detect the protein expression quantity and enzyme activity change law of caspase-3,caspase-8and caspase-9during oocytes IVM time,as well as the effects of E-64 addition.The results suggested：caspase-3,caspase-9and caspase-8protein expression quantity first increased and then decreased during oocytes IVM time,and the enzyme activity of caspase-3and caspase-9showed the same change law,while the enzyme activity of caspase-8made a difference,which kept increasing during oocytes IVMtime.Addition of E-64 had no significant influence（P 0.05）in protein expression quantity of caspase-3,caspase-8and caspase-9,and had no significant influence（P 0.05）in enzyme activity of caspase-3,but it decreased the enzyme activity of caspase-8and caspase-9（P 0.05）.The results showed that E-64 downgrades the enzyme activity of caspase-9,which plays an important role in mitochondria apoptosis pathway,and the enzyme activity of caspase-8which mediates the death receptor apoptosis pathway,therefore decreasing the apoptosis damage during oocytes IVM time.