玻璃化冷冻对猪孤雌激活囊胚中细胞凋亡的影响

发布时间：2018-05-20 01:45

本文选题：猪 + 孤雌激活胚胎　；参考：《畜牧兽医学报》2017年02期

【摘要】：旨在研究玻璃化冷冻对猪孤雌激活囊胚中细胞凋亡的影响。通过体外培养观察冷冻-解冻后囊胚腔的恢复情况,JC-1检测线粒体膜电位变化、TUNEL法检测胚胎中细胞凋亡水平,免疫荧光分析多种Caspase活性,以及qRT-PCR检测凋亡相关功能基因的mRNA表达水平。结果表明,冷冻-解冻后囊胚的囊胚腔恢复率(31.03%)和囊胚细胞数(31.81)与新鲜囊胚(100.00%和38.17)相比均显著下降(P0.05)。冷冻后囊胚的线粒体膜电位(0.46)与新鲜囊胚(1.02)相比显著下降(P0.05)。TUNEL凋亡染色后,冷冻囊胚中凋亡阳性细胞的比例(10.13%)显著高于新鲜囊胚(5.92%,P0.05)。Caspase原位荧光染色结果表明,Pan-caspase、Caspase-8、Caspase-9和Caspase-3冷冻-解冻囊胚的荧光强度值(20.65、20.60、17.58和19.88)均显著高于新鲜囊胚(7.41、6.46、5.47和6.32,P0.05)。经qRT-PCR检测,冷冻组中Caspase-8、Caspase-9、TNF-α基因的mRNA表达水平均显著高于新鲜组(P0.05),BCL-2、SOD-1的mRNA表达水平则显著低于新鲜组(P0.05)。综上表明,玻璃化冷冻可造成猪孤雌激活囊胚线粒体功能下降,并在死亡受体和线粒体途径上共同介导囊胚内细胞凋亡的发生。
[Abstract]:Aim to study the effect of vitrification on apoptosis in porcine parthenogenetic blastocysts. The recovery of blastocyst cavity after cryopreservation and thawing was observed in vitro. The changes of mitochondrial membrane potential were detected by JC-1. Apoptosis level of embryo was detected by Tunel method, and various Caspase activities were analyzed by immunofluorescence. MRNA expression level of apoptosis-related genes was detected by qRT-PCR. The results showed that the recovery rate of blastocyst (31.03) and the number of blastocysts (31.81) were significantly lower than those of fresh blastocysts (100.00% and 38.17). The mitochondrial membrane potential of frozen blastocysts (0.46) was significantly lower than that of fresh blastocysts (1.02). The percentage of apoptotic positive cells in frozen blastocysts (10.13) was significantly higher than that in fresh blastocysts (5.92%). The results of in situ fluorescence staining showed that the fluorescence intensity of Pan-caspase Caspase-8 and Caspase-3 frozen-thawed blastocysts were significantly higher than that of fresh blastocysts 7.41, 6.466.46, 5.47 and 6.32P0.05. The mRNA expression level of Caspase-8, Caspase-9, TNF- 伪 gene in frozen group was significantly higher than that in fresh group, while the mRNA expression level of BCL-2SOD-1 in fresh group was significantly lower than that in fresh group. It is concluded that vitrification can decrease the mitochondrial function of parthenogenetic blastocysts and mediate the apoptosis of blastocysts through death receptors and mitochondria pathways.
【作者单位】：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;上海海洋大学水产与生命学院;
【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)
【分类号】：S828

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