玻璃化冷冻对小鼠卵母细胞线粒体影响的初步研究

发布时间：2018-05-06 03:02

  本文选题：玻璃化冷冻 + 卵母细胞　； 参考：《广西大学》2017年硕士论文

【摘要】：为了探索玻璃化冷冻对卵母细胞线粒体的影响,从中探寻小鼠卵母细胞的玻璃化冷冻机制以及玻璃化冷冻复苏后卵母细胞受精率降低的影响因素,从而为改进玻璃化冷冻方法、提高玻璃化冷冻复苏后卵母细胞受精率提供研究思路和理论依据。本论文将研究对象分为新鲜对照组、毒性暴露组和冷冻-复苏组三组,分别通过透射电镜技术观察各组卵母细胞超微结构、生物化学发光法测量各组卵母细胞内ATP(三磷酸腺苷)含量及酶联免疫吸附法检测各组卵母细胞内线粒体膜电位、分布、ROS(活性氧族)含量和溶酶体分布情况。结果发现:1、冷冻-复苏组卵母细胞冻融后有不同程度的结构损伤,主要为:胞质中的中间纤维解体;微绒毛变短甚至缺失;线粒体粗糙模糊;胞质外围皮质颗粒数量减少;透明带变得模糊不清,有的地方甚至已经破裂;细胞基质出现大量空泡,并且形成有线粒体-空泡复合体。2、通过单因素方差分析可知,冷冻-复苏组线粒体膜电位值(0.2443±0.03819)与新鲜对照组(0.3977±0.04329)以及毒性暴露组(0.3297±0.02967)之间差异显著(P0.05),而新鲜对照组和毒性暴露组差异不显著。3、线粒体的分布情况基本上可以分为:I型完整均匀分布,Ⅱ型极化分布,Ⅲ型成群聚集分布,Ⅳ型破损缺失。其中Ⅰ型和Ⅱ型属于正常分布,Ⅲ型和Ⅳ型属于异常分布。新鲜对照组线粒体以均匀分布和极化分布的正常分布为主,分布率分别为59.54%和36.42%;毒性暴露组线粒体以均匀分布和成群分布为主,分布率分别为19.63%和69.33%;冷冻-复苏组线粒体以成群分布和破损缺失分布的异常分布为主,分布率分别为56.25%和2438%。三组卵母细胞线粒体正常分布率两两之间相比差异显著(P0.05)。4、冷冻-复苏组卵母细胞内ATP含量(0.212±0.013pmol/cell)和毒性暴露组卵母细胞内ATP含量(0.248±0.006pmol/cell)均显著性低于新鲜对照组卵母细胞(0.298±0.020pmol/cell)(P0.05)。5、冷冻-复苏组卵母细胞内ROS含量为63.81±0.94cps/枚,毒性暴露组卵母细胞内ROS含量为37.79a±1.19cps/枚,新鲜对照组卵母细胞内ROS含量为33.75±0.59cps/枚,通过单因素方差分析可知,三组卵母细胞ROS含量两两之间相比差异显著(P0.05)。6、新鲜对照组和毒性暴露组卵母细胞内溶酶体数量相对较少,且主要分布于细胞中央区域,溶酶体周围未见有未被着色的空隙;冷冻-复苏后的卵母细胞溶酶体变得大而明亮,数量很多,分布在细胞中央和周围,并且在这些大溶酶体的周围常常伴随着大量空隙。结论:玻璃化冷冻可导致小鼠卵母细胞线粒体变得粗糙模糊,线粒体膜电位值、线粒体正常分布率和ATP含量下降,ROS含量上升,溶酶体分布变得扩散。
[Abstract]:In order to explore the effect of vitrification on the mitochondria of oocytes, the mechanism of vitrification of mouse oocytes and the factors influencing the reduction of fertilization rate of oocytes after vitrification were explored in order to improve the method of vitrification. To improve the fertilization rate of vitrified cryopreservation oocytes and provide theoretical basis. In this paper, the subjects were divided into three groups: fresh control group, toxic exposure group and freeze-resuscitation group. The ultrastructure of oocytes in each group was observed by transmission electron microscopy (TEM). The contents of ATP (adenosine triphosphate) in oocytes and mitochondrial membrane potential (MMP), reactive oxygen species (Ros) content and lysosome distribution in oocytes were measured by biochemiluminescence and enzyme-linked immunosorbent assay (Elisa). The results showed that different degrees of structural damage were observed in cryopreservation and resuscitation oocytes after freezing and thawing, mainly as follows: the intermediate fibers in the cytoplasm were disintegrated, the microvilli became shorter or even absent, the mitochondria became rough and blurred, the number of cortical granules around the cytoplasm decreased. The pellucida became blurred, in some cases even ruptured, the cell matrix appeared a large number of vacuoles, and formed mtDNA-vacuole complex. 2, which was found by univariate analysis of variance (ANOVA). The mitochondrial membrane potential value of frozen-resuscitation group (0.2443 卤0.03819) was significantly different from that of fresh control group (0.3977 卤0.04329) and toxic exposure group (0.3297 卤0.02967), but there was no significant difference between fresh control group and toxic exposure group. Type complete uniform distribution, Type 鈪，

本文编号：1850475