猪MII期卵母细胞玻璃化冷冻后的凋亡途径研究  

The apoptotic pathways of vitrified porcine oocytes at MII stage

作　　者：吴彩凤[1] 戴建军[1] 钮莹芳[1] 张树山[1] 陈亚宁[1] 张德福[1] WU Cai-feng DAI Jian-jun NIU Ying-fang ZHANG Shu-shan CHEN Ya-ning ZAHNG De-fu(Division of Animal Genetic Engineering, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Animal Husbandry and Veterinary Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China)

机构地区：[1]上海市农业科学院畜牧兽医研究所,上海市农业遗传育种重点实验室动物遗传工程研究室,上海201106

出　　处：《上海农业学报》2017年第1期138-143,共6页Acta Agriculturae Shanghai

基　　金：国家自然科学基金(31372315);国家转基因生物新品种培育科技重大专项(2014ZX08006-005);上海市农业委员会青年人才计划(2014-1-32,2014-1-33)

摘　　要：为阐明猪卵母细胞玻璃化冷冻后的细胞凋亡模式,本试验利用原位荧光染色技术对冻后卵母细胞死亡受体介导的外源性凋亡途径和线粒体介导的内源性凋亡途径中的Caspase 3、Caspase 8、Caspase 9和总Caspase活性进行检测,用RT-PCR技术对不同途径中关键基因进行mRNA表达检测。结果显示:猪MII期卵母细胞冷冻后,死亡受体外源性凋亡途径的Caspase 8荧光强度值(32.03)和线粒体内源性凋亡途径的Caspase 9荧光强度值(16.56),以及两者共同途径中的Caspase 3和总Caspase荧光强度值(16.70和8.43)均显著高于新鲜卵母细胞对照组所对应的荧光强度值(分别为4.02、4.83、4.23和3.08)。死亡受体外源性凋亡途径TNFα、FasL、CASP8和CASP3基因表达量也均有一定水平的提高,线粒体介导的内源性凋亡途径中CASP9、CASP3和P53基因表达水平呈上升趋势,而Bcl2、BAX、SOD1和survivin的基因表达水平显著下降。综上所述,死亡受体介导的外源性凋亡和线粒体介导的内源性凋亡共同参与了猪MII期卵母细胞冻后的凋亡过程。In order to clarify the appopotic pathways of MII-stage porcine oocytes after vitrification, theCaspase 3, Caspase 8, Caspase 9 and Pan-caspase activities of vitrified porcine oocytes in death receptor mediatedand mitochondrion mediated apoptotic pathways were determined by in situ fluorescence staining, and the mRNAexpressions of key genes in different pathways were also detected by RT-PCR. The results were as follows：Aftervitrification of the MII-stage oocytes the fluorescence intensity values of Caspase 8, Caspase 9, Caspase 3 andPan-caspase were respectively 32.03,16.56, 16.70 and 8.43, and significantly higher than those of freshoocytes＇ control groups（being 4.02,4.83,4.23 and 3.08 respectively）. After vitrification the expression levelsof TNFa, FasL, CASP8 and CASP3 genes had a certain increasing, those of CASP9, CASP3 and P53 genespresented an upward trend,whereas those of Bcl2, BAX, SOD1 and survivin genes significantly decreased. It wasconcluded that the apoptosis of MII-stage porcine oocytes after freezing was implicated in both death receptormediated and mitochondrion mediated apoptotic pathways.

关 键 词：猪 卵母细胞 玻璃化 细胞凋亡 

分 类 号：S828[农业科学—畜牧学]