發(fā)布時間：2018-09-08 20:40

【摘要】：配子及胚胎的冷凍保存技術在人類生育能力保存和輔助生殖領域起著至關重要的作用,在家畜種質資源保存和種質資源的跨區(qū)域交流上也有著重要意義。經(jīng)過幾十年的發(fā)展,玻璃化冷凍技術有了很大提高,但冷凍造成的細胞器、細胞骨架、DNA損傷和細胞凋亡等仍是冷凍后胚胎發(fā)育能力下降的主要原因。本文利用玻璃化冷凍技術保存豬孤雌激活囊胚,檢測玻璃化冷凍過程對胚胎發(fā)育能力和凋亡水平的影響。通過在囊胚培養(yǎng)及冷凍解凍全過程添加白藜蘆醇或囊胚解凍后的孵育液中添加總Caspase抑制劑(Z-VAD-FMK),研究白藜蘆醇和總Caspase抑制劑對冷凍后的囊胚發(fā)育和凋亡水平的影響。本論文通過體外培養(yǎng)觀察冷凍解凍后囊胚腔的恢復情況,JC-1染色檢查線粒體膜電位變化、TUNEL法檢測胚胎中細胞凋亡水平,以及免疫熒光法分析多種Caspase活性,qRT-PCR檢測mRNA表達情況。結果表明,冷凍解凍后囊胚的囊胚腔恢復率和囊胚細胞數(shù)與新鮮囊胚相比均顯著下降(P0.05)、線粒體膜電位顯著降低(0.46 vs 1.02,P0.05)。TUNEL凋亡染色后,冷凍囊胚凋亡陽性細胞的比例顯著高于新鮮組(P0.05)。Caspase原位熒光染色結果表明,Pancaspase、Caspase-8、Caspase-9和Caspase-3冷凍解凍囊胚的熒光強度值均顯著高于新鮮囊胚(P0.05)。經(jīng)qRT-PCR檢測,冷凍組中Caspase-8、Caspase-9、TNF-α基因的mRNA表達水平顯著高于新鮮組,BCL-2、SOD-1的mRNA表達水平則顯著低于新鮮組(P0.05)。綜上表明,玻璃化冷凍可造成豬孤雌激活囊胚凍后線粒體功能下降,并在死亡受體和線粒體途徑上共同介導囊胚內(nèi)細胞凋亡的發(fā)生。本文利用2μmol/L RES在卵母細胞體外成熟、孤雌激活胚胎發(fā)育、玻璃化冷凍與解凍和隨后的胚胎培養(yǎng)過程中進行全程添加,觀察其對豬孤雌激活囊胚凍后線粒體膜電位、TUNEL凋亡水平、多種Caspase活性、凋亡相關功能基因mRNA表達水平和體外發(fā)育能力的影響。結果顯示:體外成熟和胚胎培養(yǎng)過程中添加RES能顯著提高卵母細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凍能力顯著提高,表現(xiàn)為囊胚腔恢復率和囊胚細胞數(shù)顯著高于冷凍組(P0.05)。RES冷凍組囊胚的線粒體膜電位顯著高于冷凍組(P0.05),但均低于新鮮囊胚。TUNEL染色顯示,兩個冷凍組中囊胚細胞發(fā)生凋亡的比例(8.24%和10.13%)要顯著高于新鮮組囊胚(5.92%,P0.05),RES添加顯著降低了囊胚中的細胞凋亡比例(P0.05)。Caspase原位熒光染色結果顯示,無論是Pan-caspase,還是Caspase-8、Caspase-9和Caspase-3,RES冷凍組的熒光強度值均顯著低于未添加RES的冷凍組(P0.05),但兩者顯著高于新鮮組。qRT-PCR檢測結果顯示,RES冷凍組Caspase-8、Caspase-9、TNF-α基因的表達量高于新鮮組,但顯著低于冷凍組(P0.05)。RES冷凍組BCL-2和SOD-1基因的表達量顯著低于新鮮組(P0.05),但是與冷凍組相比顯著升高(P0.05)。結論,RES全程添加可通過改善凍后豬孤雌激活囊胚的線粒體功能,降低囊胚細胞凋亡水平,從而提高其抗凍能力。在囊胚冷凍解凍后的孵育液中進行總Caspase抑制劑添加,觀察其對豬孤雌激活囊胚凍后線粒體膜電位、TUNEL凋亡水平、多種Caspase活性和凋亡相關功能基因mRNA表達水平的影響。結果顯示:孵育液中添加總Caspase抑制劑Z-VAD-FMK能顯著提高凍后囊胚的發(fā)育能力,表現(xiàn)為囊胚腔恢復率顯著增高和囊胚細胞數(shù)增加。與冷凍組相比,孵育液中添加Z-VAD-FMK囊胚線粒體膜電位顯著升高(P0.05)。Z-VAD-FMK組囊胚細胞凋亡比例高于新鮮組,但顯著低于冷凍組(P0.05)。Pan-caspase、Caspase-8、Caspase-9和Caspase-3原位熒光染色結果顯示,Z-VAD-FMK組的熒光強度值高于新鮮組但顯著低于冷凍組(P0.05)。qRT-PCR檢測結果顯示,Z-VAD-FMK組Caspase-8、Caspase-9、TNF-α基因的表達量高于新鮮組,但顯著低于冷凍組(P0.05)。Z-VAD-FMK組BCL-2和SOD-1基因的表達量顯著低于新鮮組(P0.05),但是與冷凍組相比顯著升高(P0.05)。結論,豬孤雌激活囊胚冷凍后孵育液中添加總Caspase抑制劑能提高線粒體膜電位水平,降低囊胚細胞凋亡水平,從而提高凍后囊胚的體外發(fā)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.
【學位授予單位】：上海海洋大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S828

【相似文獻】

相關期刊論文 前10條

1 李軍;劉兵;曾蘭;羅廷榮;;豬Caspase-3基因的克隆及序列分析[J];廣西農(nóng)業(yè)科學;2006年05期

2 袁麗杰;熊亞南;張玉琴;余利巖;張月琴;;一株具有Caspase7抑制活性的糖多孢菌屬菌株的分離鑒定[J];西南大學學報(自然科學版);2013年12期

3 黃慧;屈常林;高洪;趙汝;嚴玉霖;楊建發(fā);陳培富;鄒豐才;;內(nèi)毒素對肝臟Caspase-3蛋白表達的影響及陽離子A的保護效應[J];西南農(nóng)業(yè)學報;2013年03期

4 丁麗;方東輝;何蓮;彭文培;張燕;曾長軍;;細胞凋亡相關Caspases在豬精子冷凍過程中的表達譜研究[J];四川農(nóng)業(yè)大學學報;2014年01期

5 梁清清;石英;李衛(wèi)國;;綠茶多酚誘導肝癌細胞凋亡時Caspase-3蛋白活性與mRNA的變化[J];安徽農(nóng)業(yè)科學;2008年23期

6 高洪;屈常林;高利波;陳利平;;內(nèi)毒素血癥中肝細胞凋亡與Caspase-3蛋白表達的關系及陽離子A拮抗保護作用的研究[J];中國預防獸醫(yī)學報;2012年06期

7 程功;龔亮;陳永;胡美英;鐘國華;;家蠅細胞凋亡起始酶Caspase-1基因的克隆及在不同蟲態(tài)的表達[J];昆蟲學報;2009年07期

8 黃偉;曹錦軒;王道營;徐為民;張牧焓;;宰后鴨肉骨骼肌中caspase-3活化對嫩度的影響[J];中國農(nóng)業(yè)科學;2012年07期

9 劉楠喬;蔡亞非;許家玉;周軍智;楊帆;范睿;;體細胞Caspase-3信號通路及DNA甲基化調(diào)控卵泡閉鎖的分子機制[J];南京農(nóng)業(yè)大學學報;2010年03期

10 賈廣樂;王眾;吳紹強;林祥梅;韓雪清;劉建;梅琳;張e，

本文編號：2231637