白藜芦醇和总Caspase抑制剂对猪囊胚冷冻后凋亡和发育能力的影响
发布时间：2018-09-08 20:40
【摘要】：配子及胚胎的冷冻保存技术在人类生育能力保存和辅助生殖领域起着至关重要的作用,在家畜种质资源保存和种质资源的跨区域交流上也有着重要意义。经过几十年的发展,玻璃化冷冻技术有了很大提高,但冷冻造成的细胞器、细胞骨架、DNA损伤和细胞凋亡等仍是冷冻后胚胎发育能力下降的主要原因。本文利用玻璃化冷冻技术保存猪孤雌激活囊胚,检测玻璃化冷冻过程对胚胎发育能力和凋亡水平的影响。通过在囊胚培养及冷冻解冻全过程添加白藜芦醇或囊胚解冻后的孵育液中添加总Caspase抑制剂(Z-VAD-FMK),研究白藜芦醇和总Caspase抑制剂对冷冻后的囊胚发育和凋亡水平的影响。本论文通过体外培养观察冷冻解冻后囊胚腔的恢复情况,JC-1染色检查线粒体膜电位变化、TUNEL法检测胚胎中细胞凋亡水平,以及免疫荧光法分析多种Caspase活性,qRT-PCR检测mRNA表达情况。结果表明,冷冻解冻后囊胚的囊胚腔恢复率和囊胚细胞数与新鲜囊胚相比均显著下降(P0.05)、线粒体膜电位显著降低(0.46 vs 1.02,P0.05)。TUNEL凋亡染色后,冷冻囊胚凋亡阳性细胞的比例显著高于新鲜组(P0.05)。Caspase原位荧光染色结果表明,Pancaspase、Caspase-8、Caspase-9和Caspase-3冷冻解冻囊胚的荧光强度值均显著高于新鲜囊胚(P0.05)。经qRT-PCR检测,冷冻组中Caspase-8、Caspase-9、TNF-α基因的mRNA表达水平显著高于新鲜组,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冻后线粒体功能下降,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。本文利用2μmol/L RES在卵母细胞体外成熟、孤雌激活胚胎发育、玻璃化冷冻与解冻和随后的胚胎培养过程中进行全程添加,观察其对猪孤雌激活囊胚冻后线粒体膜电位、TUNEL凋亡水平、多种Caspase活性、凋亡相关功能基因mRNA表达水平和体外发育能力的影响。结果显示:体外成熟和胚胎培养过程中添加RES能显著提高卵母细胞孤雌激活后的卵裂率和囊胚率(P0.05)。RES全程添加后其孤雌激活囊胚的抗冻能力显著提高,表现为囊胚腔恢复率和囊胚细胞数显著高于冷冻组(P0.05)。RES冷冻组囊胚的线粒体膜电位显著高于冷冻组(P0.05),但均低于新鲜囊胚。TUNEL染色显示,两个冷冻组中囊胚细胞发生凋亡的比例(8.24%和10.13%)要显著高于新鲜组囊胚(5.92%,P0.05),RES添加显著降低了囊胚中的细胞凋亡比例(P0.05)。Caspase原位荧光染色结果显示,无论是Pan-caspase,还是Caspase-8、Caspase-9和Caspase-3,RES冷冻组的荧光强度值均显著低于未添加RES的冷冻组(P0.05),但两者显著高于新鲜组。qRT-PCR检测结果显示,RES冷冻组Caspase-8、Caspase-9、TNF-α基因的表达量高于新鲜组,但显著低于冷冻组(P0.05)。RES冷冻组BCL-2和SOD-1基因的表达量显著低于新鲜组(P0.05),但是与冷冻组相比显著升高(P0.05)。结论,RES全程添加可通过改善冻后猪孤雌激活囊胚的线粒体功能,降低囊胚细胞凋亡水平,从而提高其抗冻能力。在囊胚冷冻解冻后的孵育液中进行总Caspase抑制剂添加,观察其对猪孤雌激活囊胚冻后线粒体膜电位、TUNEL凋亡水平、多种Caspase活性和凋亡相关功能基因mRNA表达水平的影响。结果显示:孵育液中添加总Caspase抑制剂Z-VAD-FMK能显著提高冻后囊胚的发育能力,表现为囊胚腔恢复率显著增高和囊胚细胞数增加。与冷冻组相比,孵育液中添加Z-VAD-FMK囊胚线粒体膜电位显著升高(P0.05)。Z-VAD-FMK组囊胚细胞凋亡比例高于新鲜组,但显著低于冷冻组(P0.05)。Pan-caspase、Caspase-8、Caspase-9和Caspase-3原位荧光染色结果显示,Z-VAD-FMK组的荧光强度值高于新鲜组但显著低于冷冻组(P0.05)。qRT-PCR检测结果显示,Z-VAD-FMK组Caspase-8、Caspase-9、TNF-α基因的表达量高于新鲜组,但显著低于冷冻组(P0.05)。Z-VAD-FMK组BCL-2和SOD-1基因的表达量显著低于新鲜组(P0.05),但是与冷冻组相比显著升高(P0.05)。结论,猪孤雌激活囊胚冷冻后孵育液中添加总Caspase抑制剂能提高线粒体膜电位水平,降低囊胚细胞凋亡水平,从而提高冻后囊胚的体外发育能力。
[Abstract]:Cryopreservation of gametes and embryos plays an important role in the field of human fertility conservation and assisted reproduction. It is also of great significance in the preservation of livestock germplasm resources and the cross-regional exchange of germplasm resources. DNA damage and apoptosis are still the main reasons for the decline of embryo development ability after cryopreservation. * vitrification technology was used to preserve Parthenogenetic Activated blastocysts in pigs, and the effects of vitrification on embryo development ability and apoptosis level were detected. The effects of total caspase inhibitors (Z-VAD-FMK) and resveratrol on the development and apoptosis of frozen blastocysts were studied by adding total caspase inhibitors (Z-VAD-FMK). The results showed that the recovery rate of blastocyst lumen and the number of blastocyst cells in frozen-thawed blastocysts were significantly lower than those in fresh blastocysts (P 0.05), and the mitochondrial membrane potential was significantly lower (0.46 vs 1.02, P 0.05). Caspase in situ fluorescence staining showed that the fluorescence intensity of Pancaspase, Caspase-8, Caspase-9 and Caspase-3 frozen-thawed blastocysts was significantly higher than that of fresh blastocysts (P 0.05). The expression of Caspase-8, Caspase-9 and TNF-alpha mRNA in frozen blastocysts was significantly higher than that in fresh blastocysts (P 0.05). The expression level of mRNA in SOD-1 was significantly lower than that in the fresh group (P0.05). * it is concluded that vitrification can induce mitochondrial function decline after parthenogenetic activation of blastocysts, and mediate apoptosis in blastocysts on the death receptor and mitochondrial pathway. In this paper, 2 mol/L RES is used to maturation in vitro, and parthenogenetic activation of the embryo. During the vitrification and thawing and subsequent embryo culture, the mitochondrial membrane potential, TUNEL * apoptosis, Caspase activity, mRNA expression level and in vitro development ability of the Parthenogenetic Activated blastocysts after cryopreservation were observed. RES could significantly increase the cleavage rate and blastocyst rate (P 0.05). The freezing resistance of parthenogenetically activated blastocysts was significantly improved with the addition of RES. The recovery rate of blastocyst cavity and the number of blastocysts were significantly higher in the RES freezing group than in the cryopreservation group (P 0.05). The mitochondrial membrane potential of blastocysts in the RES freezing group was significantly higher than that in the cryopreservation group (P 0.05), but both were lower. TUNEL staining showed that the apoptosis rate of blastocysts in the two cryopreservation groups (8.24% and 10.13%) was significantly higher than that in the fresh blastocysts (5.92%, P 0.05). RES addition significantly reduced the apoptosis rate of blastocysts (P 0.05). Caspase in situ fluorescence staining showed that both Pan-caspase and Caspase-8, Caspase-9 and Caspase-8, Caspase-9 and Caspa were significantly lower than those in the fresh blastocysts (P 0.05). The fluorescence intensity values of se-3 and RES frozen group were significantly lower than those of non-RES frozen group (P 0.05), but both of them were significantly higher than those of fresh group. The fresh group (P0.05), but significantly increased (P0.05) compared with the frozen group. Conclusion * the whole process of RES can increase the mitochondrial function of the Parthenogenetic Activated blastocysts and reduce the apoptosis level of blastocyst cells, thereby enhancing its antifreeze ability. The total Caspase inhibitor was added to the incubated * frozen thawed frozen thawed broth to observe its parthenogenetic stimulation. The results showed that the total caspase inhibitor Z-VAD-FMK in the incubator could significantly improve the development ability of frozen blastocysts. The recovery rate of blastocyst cavity and the number of blastocyst cells increased significantly. The mitochondrial membrane potential of Z-VAD-FMK group was significantly higher than that of the fresh group (P 0.05). The apoptosis rate of Z-VAD-FMK group was significantly higher than that of the frozen group (P 0.05). Pan-caspase, Caspase-8, Caspase-9 and Caspase-3 in situ fluorescence staining showed that the fluorescence intensity of Z-VAD-FMK group was higher than that of the fresh group but significantly lower than that of the frozen group (P 0.05). Frozen group (P0.05).QRT-PCR test showed that the expression level of Caspase-8, Caspase-9 and TNF- alpha genes in Z-VAD-FMK group was higher than that in fresh group, but significantly lower than that in frozen group (P0.05).Z-VAD-FMK group BCL-2 and SOD-1 gene expression was significantly lower than that in fresh group (P0.05), but significantly increased (*) compared with cold group. Addition of total caspase inhibitors in post-incubation broth can increase the level of mitochondrial membrane potential and decrease the level of apoptosis of blastocysts, thus improving the in vitro development of frozen blastocysts.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828
[Abstract]:Cryopreservation of gametes and embryos plays an important role in the field of human fertility conservation and assisted reproduction. It is also of great significance in the preservation of livestock germplasm resources and the cross-regional exchange of germplasm resources. DNA damage and apoptosis are still the main reasons for the decline of embryo development ability after cryopreservation. * vitrification technology was used to preserve Parthenogenetic Activated blastocysts in pigs, and the effects of vitrification on embryo development ability and apoptosis level were detected. The effects of total caspase inhibitors (Z-VAD-FMK) and resveratrol on the development and apoptosis of frozen blastocysts were studied by adding total caspase inhibitors (Z-VAD-FMK). The results showed that the recovery rate of blastocyst lumen and the number of blastocyst cells in frozen-thawed blastocysts were significantly lower than those in fresh blastocysts (P 0.05), and the mitochondrial membrane potential was significantly lower (0.46 vs 1.02, P 0.05). Caspase in situ fluorescence staining showed that the fluorescence intensity of Pancaspase, Caspase-8, Caspase-9 and Caspase-3 frozen-thawed blastocysts was significantly higher than that of fresh blastocysts (P 0.05). The expression of Caspase-8, Caspase-9 and TNF-alpha mRNA in frozen blastocysts was significantly higher than that in fresh blastocysts (P 0.05). The expression level of mRNA in SOD-1 was significantly lower than that in the fresh group (P0.05). * it is concluded that vitrification can induce mitochondrial function decline after parthenogenetic activation of blastocysts, and mediate apoptosis in blastocysts on the death receptor and mitochondrial pathway. In this paper, 2 mol/L RES is used to maturation in vitro, and parthenogenetic activation of the embryo. During the vitrification and thawing and subsequent embryo culture, the mitochondrial membrane potential, TUNEL * apoptosis, Caspase activity, mRNA expression level and in vitro development ability of the Parthenogenetic Activated blastocysts after cryopreservation were observed. RES could significantly increase the cleavage rate and blastocyst rate (P 0.05). The freezing resistance of parthenogenetically activated blastocysts was significantly improved with the addition of RES. The recovery rate of blastocyst cavity and the number of blastocysts were significantly higher in the RES freezing group than in the cryopreservation group (P 0.05). The mitochondrial membrane potential of blastocysts in the RES freezing group was significantly higher than that in the cryopreservation group (P 0.05), but both were lower. TUNEL staining showed that the apoptosis rate of blastocysts in the two cryopreservation groups (8.24% and 10.13%) was significantly higher than that in the fresh blastocysts (5.92%, P 0.05). RES addition significantly reduced the apoptosis rate of blastocysts (P 0.05). Caspase in situ fluorescence staining showed that both Pan-caspase and Caspase-8, Caspase-9 and Caspase-8, Caspase-9 and Caspa were significantly lower than those in the fresh blastocysts (P 0.05). The fluorescence intensity values of se-3 and RES frozen group were significantly lower than those of non-RES frozen group (P 0.05), but both of them were significantly higher than those of fresh group. The fresh group (P0.05), but significantly increased (P0.05) compared with the frozen group. Conclusion * the whole process of RES can increase the mitochondrial function of the Parthenogenetic Activated blastocysts and reduce the apoptosis level of blastocyst cells, thereby enhancing its antifreeze ability. The total Caspase inhibitor was added to the incubated * frozen thawed frozen thawed broth to observe its parthenogenetic stimulation. The results showed that the total caspase inhibitor Z-VAD-FMK in the incubator could significantly improve the development ability of frozen blastocysts. The recovery rate of blastocyst cavity and the number of blastocyst cells increased significantly. The mitochondrial membrane potential of Z-VAD-FMK group was significantly higher than that of the fresh group (P 0.05). The apoptosis rate of Z-VAD-FMK group was significantly higher than that of the frozen group (P 0.05). Pan-caspase, Caspase-8, Caspase-9 and Caspase-3 in situ fluorescence staining showed that the fluorescence intensity of Z-VAD-FMK group was higher than that of the fresh group but significantly lower than that of the frozen group (P 0.05). Frozen group (P0.05).QRT-PCR test showed that the expression level of Caspase-8, Caspase-9 and TNF- alpha genes in Z-VAD-FMK group was higher than that in fresh group, but significantly lower than that in frozen group (P0.05).Z-VAD-FMK group BCL-2 and SOD-1 gene expression was significantly lower than that in fresh group (P0.05), but significantly increased (*) compared with cold group. Addition of total caspase inhibitors in post-incubation broth can increase the level of mitochondrial membrane potential and decrease the level of apoptosis of blastocysts, thus improving the in vitro development of frozen blastocysts.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828
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