液氦玻璃化冷冻对牛未成熟卵母细胞超微结构和相关基因表达的影响

发布时间：2018-02-13 20:23

本文关键词： 牛未成熟卵母细胞玻璃化冷冻液氦超微结构基因表达　出处：《河南科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：未成熟卵母细胞低温保存对动物种质资源的保护有重要意义。虽然有些成功的报道,但因冷冻过程中造成的结构和功能损伤,使其发育能力仍不理想。为此,我们建立了一种以液氦(LHe,㧟269°C)为冷源进行玻璃化冷冻牛未成熟卵母细胞的方法。目前的研究表明:液氦玻璃化冷冻法在提高冷冻速率的同时,也降低了冷冻保护剂的浓度。然而,该方法是否会对卵母细胞的形态结构和相关基因表达造成损伤还不清楚。本研究的目的是探讨液氦OPS玻璃化冷冻对牛未成熟卵母细胞发育能力、超微结构和相关基因表达的影响。以下研究,均以牛未成熟的卵母细胞为实验材料,随机分为新鲜组(Control组,阴性对照)、液氮冷冻处理组(LN组,阳性对照)和液氦处理组(LHe组)。实验1:液氦玻璃化冷冻对牛未成熟卵母细胞体外发育能力的影响。液氦玻璃化冷冻牛未成熟卵母细胞,然后将其解冻并进行体外培养,统计其形态正常率、成熟率、卵裂率和囊胚率等发育指标变化,分析液氦冷冻的效果。液氦组的各项发育指标(87.1%、51%、41.7%和13%)显著高于液氮组(80.5%、48%、36.8%和8.5%,P0.05),但冷冻组的发育能力均低于新鲜对照组(100%、73.2%、62%和39.8%,P0.05)。结果表明,液氦冷冻牛未成熟卵母细胞的方法,改善了冷冻后卵母细胞的发育能力。实验2:液氦玻璃化冷冻对牛未成熟卵母细胞超微结构的影响。借助透射电子显微镜设备,观察液氦玻璃化冷冻牛未成熟卵母细胞后,其透明带、线粒体、皮质颗粒和脂滴等超微结构的变化。液氦组卵母细胞的超微结构损伤明显小于液氮处理组,表现在线粒体的数量与完整度、皮质颗粒的数量与分布以及脂滴含量较少等方面。但两个冷冻组明显不如对照组的结构清晰而完整,主要表现为卵周隙和微绒毛基本消失、细胞内基质密度小而模糊及线粒体不同程度的退化等方面。这些结果说明,液氦冷冻牛未成熟卵母细胞的过程降低了液氮对其超微结构的损伤程度。实验3:液氦玻璃化冷冻对牛未成熟卵母细胞体相关基因表达的影响。采用实时荧光定量PCR的方法检测液氦玻璃化冷冻牛未成熟卵母细胞后,卵母细胞内P53、CDC20、Eg5和Npm2等与发育相关基因mRNA表达量的变化。P53和Eg5两个基因,在液氮组中的表达水平显著高于液氦组和对照组(P0.05),且P53基因在液氦组和对照组之间差异不显著(P0.05)。液氮组和液氦组的CDC20基因表达量差异不显著(P0.05),但两者均显著低于对照组。Npm2基因在液氦组的表达量低于对照组(P0.05),但液氮组和对照组之间差异不显著(P0.05)。因此,液氦冷冻明显改善了液氮冷冻对凋亡基因P53和驱动蛋白Eg5的影响。本研究中液氦玻璃化冷冻牛未成熟卵母细胞的方法,降低了低温损伤对卵母细胞细胞器超微结构和一些相关基因表达的负面影响,提高了卵母细胞后期发育的能力,从而为未成熟卵母细胞冷冻方法的探索提供一定的参考依据。
[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 掳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.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823

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