玻璃化冷冻对猪孤雌激活囊胚中细胞凋亡的影响

doi:10.11843/j.issn.0366-6964.2017.02.007

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (2): 243-251.doi: 10.11843/j.issn.0366-6964.2017.02.007

玻璃化冷冻对猪孤雌激活囊胚中细胞凋亡的影响

陈亚宁^1,2,3，吴彩凤^1,2*，戴建军^1,2，张树山^1,2，钮莹芳^1,2,3，张德福^1,2*

（1.上海市农业科学院畜牧兽医研究所，上海 201106；2.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；3.上海海洋大学水产与生命学院，上海 201306）

收稿日期:2016-07-20 出版日期:2017-02-23 发布日期:2017-02-23
通讯作者: 张德福，E-mail：zhangdefuzdf@163.com；吴彩凤，E-mail：wucaifengwcf@163.com
作者简介:陈亚宁(1990-)，男，安徽宿州人，硕士生，主要从事动物胚胎工程研究，E-mail：chenyaning1102@hotmail.com
基金资助:
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）

Effect of Vitrification on Cell Apoptotic Levels of Porcine Parthenogenetic Blastocysts

CHEN Ya-ning^1,2,3, WU Cai-feng^1,2* , DAI Jian-jun^1,2, ZHANG Shu-shan^1,2, NIU Ying-fang^1,2,3, ZHANG De-fu^1,2*

(1.Institute of Animal Science and Veterinary Medicine, Shanghai Academy of Agricultural Sciences,Shanghai 201106, China; 2. Division of Animal Genetic Engineering, Shanghai Municipal Key Laboratory of Agri-genetics and Breeding, Shanghai 201106, China; 3. College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China)

Received:2016-07-20 Online:2017-02-23 Published:2017-02-23

摘要/Abstract

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（P＜0.05）。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（P＜0.05）。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%，P＜0.05）。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，P＜0.05）。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（P＜0.05），BCL-2、SOD-1的mRNA表达水平则显著低于新鲜组（P＜0.05）。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。

关键词: 猪, 孤雌激活胚胎, 玻璃化冷冻, 线粒体, 凋亡, 凋亡途径

Abstract:

The objective of this study was to evaluate the effect of vitrification on cell apoptotic levels of porcine parthenogenetic blastocysts. The blastocoel recovery, mitochondrial membrane potential (ΔΨm), early apoptotic levels and the activities of several caspases of frozen thawed blastocysts were measured, and the mRNA expression levels of apoptosis related genes involved in different apoptotic pathways were also measured. The results showed that the rate of re-expanded blastocysts (31.03%) and total cells (31.81) of vitrified blastocysts were significantly lower than those of fresh group (100.00% and 38.17, P<0.05). The mean mitochondrial ΔΨm of vitrification group was 0.46, which was significantly lower than that of fresh group (1.02, P＜0.05). TUNEL assay revealed a significantly higher rate of DNA fragmentation in the cells of vitrified blastocysts (10.13%), which was significantly higher than that of fresh group (5.92%，P＜0.05). This study also demonstrated that vitrification caused a significant increasing of Pan-caspase, Caspase-3, Caspase-8 and Caspase-9 activities in vitrified group (20.65, 20.60, 17.58 and 19.88) comparing with fresh group (7.41, 6.46, 5.47 and 6.32, P＜0.05). qRT-PCR results showed that the relative expression levels of pro-apoptotic genes (Caspase-8, Caspase-9, TNF-α) were up-regulated and the relative expression of anti-apoptotic genes (Bcl-2 and SOD-1) were down-regulated greatly in vitrified group (P＜0.05). This study concluded that the in vitro developmental competence of blastocysts after vitrification was reduced by the decreasing of mitochondrial function, and the increasing of apoptotic levels were mediated by both death receptor and mitochondria apoptotic pathways.

Key words: porcine, parthenogenetic embryo, vitrification, mitochondria, apoptosis, apoptotic pathway

中图分类号:

S828.2

陈亚宁，吴彩凤，戴建军，张树山，钮莹芳，张德福. 玻璃化冷冻对猪孤雌激活囊胚中细胞凋亡的影响[J]. 畜牧兽医学报, 2017, 48(2): 243-251.

CHEN Ya-ning, WU Cai-feng, DAI Jian-jun, ZHANG Shu-shan, NIU Ying-fang, ZHANG De-fu. Effect of Vitrification on Cell Apoptotic Levels of Porcine Parthenogenetic Blastocysts[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(2): 243-251.

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玻璃化冷冻对猪孤雌激活囊胚中细胞凋亡的影响

Effect of Vitrification on Cell Apoptotic Levels of Porcine Parthenogenetic Blastocysts

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