發(fā)布時(shí)間：2018-05-06 03:02

  本文選題：玻璃化冷凍 + 卵母細(xì)胞��； 參考：《廣西大學(xué)》2017年碩士論文

【摘要】：為了探索玻璃化冷凍對(duì)卵母細(xì)胞線粒體的影響,從中探尋小鼠卵母細(xì)胞的玻璃化冷凍機(jī)制以及玻璃化冷凍復(fù)蘇后卵母細(xì)胞受精率降低的影響因素,從而為改進(jìn)玻璃化冷凍方法、提高玻璃化冷凍復(fù)蘇后卵母細(xì)胞受精率提供研究思路和理論依據(jù)。本論文將研究對(duì)象分為新鮮對(duì)照組、毒性暴露組和冷凍-復(fù)蘇組三組,分別通過(guò)透射電鏡技術(shù)觀察各組卵母細(xì)胞超微結(jié)構(gòu)、生物化學(xué)發(fā)光法測(cè)量各組卵母細(xì)胞內(nèi)ATP(三磷酸腺苷)含量及酶聯(lián)免疫吸附法檢測(cè)各組卵母細(xì)胞內(nèi)線粒體膜電位、分布、ROS(活性氧族)含量和溶酶體分布情況。結(jié)果發(fā)現(xiàn):1、冷凍-復(fù)蘇組卵母細(xì)胞凍融后有不同程度的結(jié)構(gòu)損傷,主要為:胞質(zhì)中的中間纖維解體;微絨毛變短甚至缺失;線粒體粗糙模糊;胞質(zhì)外圍皮質(zhì)顆粒數(shù)量減少;透明帶變得模糊不清,有的地方甚至已經(jīng)破裂;細(xì)胞基質(zhì)出現(xiàn)大量空泡,并且形成有線粒體-空泡復(fù)合體。2、通過(guò)單因素方差分析可知,冷凍-復(fù)蘇組線粒體膜電位值(0.2443±0.03819)與新鮮對(duì)照組(0.3977±0.04329)以及毒性暴露組(0.3297±0.02967)之間差異顯著(P0.05),而新鮮對(duì)照組和毒性暴露組差異不顯著。3、線粒體的分布情況基本上可以分為:I型完整均勻分布,Ⅱ型極化分布,Ⅲ型成群聚集分布,Ⅳ型破損缺失。其中Ⅰ型和Ⅱ型屬于正常分布,Ⅲ型和Ⅳ型屬于異常分布。新鮮對(duì)照組線粒體以均勻分布和極化分布的正常分布為主,分布率分別為59.54%和36.42%;毒性暴露組線粒體以均勻分布和成群分布為主,分布率分別為19.63%和69.33%;冷凍-復(fù)蘇組線粒體以成群分布和破損缺失分布的異常分布為主,分布率分別為56.25%和2438%。三組卵母細(xì)胞線粒體正常分布率兩兩之間相比差異顯著(P0.05)。4、冷凍-復(fù)蘇組卵母細(xì)胞內(nèi)ATP含量(0.212±0.013pmol/cell)和毒性暴露組卵母細(xì)胞內(nèi)ATP含量(0.248±0.006pmol/cell)均顯著性低于新鮮對(duì)照組卵母細(xì)胞(0.298±0.020pmol/cell)(P0.05)。5、冷凍-復(fù)蘇組卵母細(xì)胞內(nèi)ROS含量為63.81±0.94cps/枚,毒性暴露組卵母細(xì)胞內(nèi)ROS含量為37.79a±1.19cps/枚,新鮮對(duì)照組卵母細(xì)胞內(nèi)ROS含量為33.75±0.59cps/枚,通過(guò)單因素方差分析可知,三組卵母細(xì)胞ROS含量?jī)蓛芍g相比差異顯著(P0.05)。6、新鮮對(duì)照組和毒性暴露組卵母細(xì)胞內(nèi)溶酶體數(shù)量相對(duì)較少,且主要分布于細(xì)胞中央?yún)^(qū)域,溶酶體周?chē)匆?jiàn)有未被著色的空隙;冷凍-復(fù)蘇后的卵母細(xì)胞溶酶體變得大而明亮,數(shù)量很多,分布在細(xì)胞中央和周?chē)?并且在這些大溶酶體的周?chē)３０殡S著大量空隙。結(jié)論:玻璃化冷凍可導(dǎo)致小鼠卵母細(xì)胞線粒體變得粗糙模糊,線粒體膜電位值、線粒體正常分布率和ATP含量下降,ROS含量上升,溶酶體分布變得擴(kuò)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 鹵0.03819) was significantly different from that of fresh control group (0.3977 鹵0.04329) and toxic exposure group (0.3297 鹵0.02967), but there was no significant difference between fresh control group and toxic exposure group. Type complete uniform distribution, Type 鈪，

本文編號(hào)：1850475