本文關(guān)鍵詞： 牛未成熟卵母細(xì)胞 玻璃化冷凍 液氦 超微結(jié)構(gòu) 基因表達(dá)　出處：《河南科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：未成熟卵母細(xì)胞低溫保存對(duì)動(dòng)物種質(zhì)資源的保護(hù)有重要意義。雖然有些成功的報(bào)道,但因冷凍過(guò)程中造成的結(jié)構(gòu)和功能損傷,使其發(fā)育能力仍不理想。為此,我們建立了一種以液氦(LHe,㧟269°C)為冷源進(jìn)行玻璃化冷凍牛未成熟卵母細(xì)胞的方法。目前的研究表明:液氦玻璃化冷凍法在提高冷凍速率的同時(shí),也降低了冷凍保護(hù)劑的濃度。然而,該方法是否會(huì)對(duì)卵母細(xì)胞的形態(tài)結(jié)構(gòu)和相關(guān)基因表達(dá)造成損傷還不清楚。本研究的目的是探討液氦OPS玻璃化冷凍對(duì)牛未成熟卵母細(xì)胞發(fā)育能力、超微結(jié)構(gòu)和相關(guān)基因表達(dá)的影響。以下研究,均以牛未成熟的卵母細(xì)胞為實(shí)驗(yàn)材料,隨機(jī)分為新鮮組(Control組,陰性對(duì)照)、液氮冷凍處理組(LN組,陽(yáng)性對(duì)照)和液氦處理組(LHe組)。實(shí)驗(yàn)1:液氦玻璃化冷凍對(duì)牛未成熟卵母細(xì)胞體外發(fā)育能力的影響。液氦玻璃化冷凍牛未成熟卵母細(xì)胞,然后將其解凍并進(jìn)行體外培養(yǎng),統(tǒng)計(jì)其形態(tài)正常率、成熟率、卵裂率和囊胚率等發(fā)育指標(biāo)變化,分析液氦冷凍的效果。液氦組的各項(xiàng)發(fā)育指標(biāo)(87.1%、51%、41.7%和13%)顯著高于液氮組(80.5%、48%、36.8%和8.5%,P0.05),但冷凍組的發(fā)育能力均低于新鮮對(duì)照組(100%、73.2%、62%和39.8%,P0.05)。結(jié)果表明,液氦冷凍牛未成熟卵母細(xì)胞的方法,改善了冷凍后卵母細(xì)胞的發(fā)育能力。實(shí)驗(yàn)2:液氦玻璃化冷凍對(duì)牛未成熟卵母細(xì)胞超微結(jié)構(gòu)的影響。借助透射電子顯微鏡設(shè)備,觀察液氦玻璃化冷凍牛未成熟卵母細(xì)胞后,其透明帶、線粒體、皮質(zhì)顆粒和脂滴等超微結(jié)構(gòu)的變化。液氦組卵母細(xì)胞的超微結(jié)構(gòu)損傷明顯小于液氮處理組,表現(xiàn)在線粒體的數(shù)量與完整度、皮質(zhì)顆粒的數(shù)量與分布以及脂滴含量較少等方面。但兩個(gè)冷凍組明顯不如對(duì)照組的結(jié)構(gòu)清晰而完整,主要表現(xiàn)為卵周隙和微絨毛基本消失、細(xì)胞內(nèi)基質(zhì)密度小而模糊及線粒體不同程度的退化等方面。這些結(jié)果說(shuō)明,液氦冷凍牛未成熟卵母細(xì)胞的過(guò)程降低了液氮對(duì)其超微結(jié)構(gòu)的損傷程度。實(shí)驗(yàn)3:液氦玻璃化冷凍對(duì)牛未成熟卵母細(xì)胞體相關(guān)基因表達(dá)的影響。采用實(shí)時(shí)熒光定量PCR的方法檢測(cè)液氦玻璃化冷凍牛未成熟卵母細(xì)胞后,卵母細(xì)胞內(nèi)P53、CDC20、Eg5和Npm2等與發(fā)育相關(guān)基因mRNA表達(dá)量的變化。P53和Eg5兩個(gè)基因,在液氮組中的表達(dá)水平顯著高于液氦組和對(duì)照組(P0.05),且P53基因在液氦組和對(duì)照組之間差異不顯著(P0.05)。液氮組和液氦組的CDC20基因表達(dá)量差異不顯著(P0.05),但兩者均顯著低于對(duì)照組。Npm2基因在液氦組的表達(dá)量低于對(duì)照組(P0.05),但液氮組和對(duì)照組之間差異不顯著(P0.05)。因此,液氦冷凍明顯改善了液氮冷凍對(duì)凋亡基因P53和驅(qū)動(dòng)蛋白Eg5的影響。本研究中液氦玻璃化冷凍牛未成熟卵母細(xì)胞的方法,降低了低溫?fù)p傷對(duì)卵母細(xì)胞細(xì)胞器超微結(jié)構(gòu)和一些相關(guān)基因表達(dá)的負(fù)面影響,提高了卵母細(xì)胞后期發(fā)育的能力,從而為未成熟卵母細(xì)胞冷凍方法的探索提供一定的參考依據(jù)。
[Abstract]:Cryopreservation of immature oocytes is of great significance to the protection of animal germplasm resources. Although some successful reports have been reported, the developmental ability of immature oocytes is still not satisfactory due to the structural and functional damage caused by freezing. We have created a liquid helium LHee? 269 擄C) as a cold source for vitrification of bovine immature oocytes. Current studies have shown that liquid helium vitrification not only increases the freezing rate but also reduces the concentration of cryopreservation agents. It is not clear whether this method will damage the morphology and related gene expression of oocytes. The purpose of this study was to investigate the developmental ability of bovine immature oocytes by vitrification of liquid helium OPS. The following studies were conducted on immature bovine oocytes as experimental materials, which were randomly divided into fresh control group, negative control group, liquid nitrogen cryopreservation group and LN group. Experiment 1: effect of vitrification of liquid helium on the development of bovine immature oocytes in vitro. Liquid helium vitrified bovine immature oocytes were frozen, thawed and cultured in vitro. The changes of morphological normal rate, maturation rate, cleavage rate and blastocyst rate were calculated. The effects of liquid helium freezing were analyzed. The developmental indexes of liquid helium group were significantly higher than that of liquid nitrogen group (80.5%, 36.8% and 8.5%, P 0.05). However, the developmental capacity of the freezing group was lower than that of the fresh control group (73.262% and 39.8%, P 0.05). The results showed that the liquid helium cryopreservation was the method of immature bovine oocytes. The effect of vitrification of liquid helium on the ultrastructure of immature bovine oocytes was studied by means of transmission electron microscope (TEM), and the effect of vitrification of liquid helium on the ultrastructure of immature bovine oocytes was observed. The ultrastructural changes of pellucida, mitochondria, cortical granules and lipid droplets. The ultrastructural damage of oocytes in liquid helium group was significantly less than that in liquid nitrogen treatment group, which was manifested in the number and integrity of mitochondria. The number and distribution of cortical granules and the content of lipid droplets were less, but the structure of the two freezing groups was not as clear and complete as that of the control group, mainly showing the disappearance of periegg space and microvilli. These results suggest that the density of intracellular matrix is small and blurred, and mitochondria degenerate to varying degrees. The process of freezing immature bovine oocytes with liquid helium reduced the damage of liquid nitrogen to its ultrastructure. Experiment 3: effect of liquid helium vitrification on the expression of genes related to bovine immature oocytes. After cryopreservation of bovine immature oocytes with liquid helium vitrified by PCR, The changes of mRNA expression of developmental related genes, such as P53, CDC20, Eg5 and Npm2, in oocytes. P53 and Eg5 genes. The expression level of p53 gene in liquid nitrogen group was significantly higher than that in liquid helium group and control group, and there was no significant difference in p53 gene expression between liquid helium group and control group. There was no significant difference in CDC20 gene expression between liquid nitrogen group and liquid helium group, but both of them were significantly lower than that in liquid helium group. The expression of Npm2 gene in the liquid helium group was lower than that in the control group, but there was no significant difference between the liquid nitrogen group and the control group. Liquid helium cryopreservation significantly improved the effect of liquid nitrogen freezing on apoptotic gene p53 and Eg5. It reduced the negative effects of hypothermia injury on the ultrastructure of organelles and the expression of some related genes in oocytes, and improved the ability of oocyte development in the later stage, thus providing a certain reference for the exploration of cryopreservation methods for immature oocytes.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S823

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