發(fā)布時(shí)間：2018-03-01 01:27

  本文關(guān)鍵詞： 小鼠 Ezrin 卵母細(xì)胞 體外成熟　出處：《安徽農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：Ezrin是ERM蛋白家族一員,在多種細(xì)胞中橋接細(xì)胞骨架與質(zhì)膜,參與細(xì)胞形態(tài)維持、細(xì)胞粘附、細(xì)胞遷移及信號(hào)轉(zhuǎn)導(dǎo)等多種細(xì)胞功能。有研究表明該家族蛋白參與了小鼠卵母細(xì)胞皮層緊張度的調(diào)控及第二次成熟分裂時(shí)紡錘體的定位。我們前期的研究發(fā)現(xiàn),小鼠GV期到MII期的卵母細(xì)胞均有Ezrin及其磷酸化修飾的活性形式存在,但其在小鼠卵母細(xì)胞成熟過程究竟發(fā)揮什么作用尚不清楚。本試驗(yàn)以小鼠GV期裸卵為材料,通過顯微注射特異性siRNA敲低Ezrin的方法研究了該蛋白在小鼠卵母細(xì)胞發(fā)育成熟中的作用以及對(duì)受精后胚胎發(fā)育潛力的影響。研究?jī)?nèi)容與結(jié)果如下:1.顯微注射Ezrin-siRNA后不同時(shí)間小鼠GV期裸卵內(nèi)Ezrin的表達(dá)水平向小鼠GV期裸卵顯微注射Ezrin-siRNA或NC-siRNA(陰性對(duì)照),在含Milrinone(抑制細(xì)胞核分裂)M2中分別抑制0 h、6 h、12 h、18 h、24 h、30h和36 h后,用Ezrin特異性抗體進(jìn)行熒光染色并用Image J軟件分析所獲圖片的平均熒光值。結(jié)果發(fā)現(xiàn):在顯微注射后18~30 h期間,試驗(yàn)組Ezrin表達(dá)量顯著低于對(duì)照組(P0.05)。2.Milrinone阻滯不同時(shí)間對(duì)小鼠GV期裸卵體外成熟率的影響將小鼠GV期裸卵在Milrinone培養(yǎng)液中分別阻滯0 h(對(duì)照組)、6 h、12 h、18 h、24 h、30 h和36 h后,統(tǒng)計(jì)各組卵母細(xì)胞成熟率(以排出PBI計(jì))。結(jié)果顯示,阻滯時(shí)間≤18 h,各組卵母細(xì)胞成熟率與對(duì)照組無顯著差異(P0.05);阻滯時(shí)間≥24 h,則卵母細(xì)胞成熟率顯著低于對(duì)照組及阻滯時(shí)間≤18 h的各試驗(yàn)組(P0.05)。3.敲低Ezrin對(duì)小鼠卵母細(xì)胞細(xì)胞體外成熟的影響向小鼠GV期裸卵中顯微注射Ezrin-siRNA或NC-siRNA(陰性對(duì)照)后,Milrinone阻滯18 h再繼續(xù)成熟培養(yǎng),結(jié)果發(fā)現(xiàn):敲低Ezrin蛋白后,小鼠卵母細(xì)胞體外成熟率顯著低于對(duì)照組(P0.05);皮層顆粒(cortical granule,CGs)異常分布率及MII期紡錘體位置異常率顯著高于對(duì)照組(P0.05);表面微絨毛長(zhǎng)度顯著降低(P0.05),密度極顯著降低(P0.01)。4.敲低Ezrin后對(duì)小鼠卵母細(xì)胞IVF的影響分別取試驗(yàn)組(GV期注射Ezrin-siRNA)和對(duì)照組(GV期注射NC-siRNA)所獲得的小鼠MII期卵母細(xì)胞進(jìn)行體外受精(IVF),結(jié)果發(fā)現(xiàn):與對(duì)照組相比,GV期敲低Ezrin導(dǎo)致小鼠成熟卵母細(xì)胞受精率有下降(51.34±4.94%),不過差異不顯著(P0.05),但試驗(yàn)組的囊胚率極顯著低于對(duì)照組(P0.01)。
[Abstract]:Ezrin is a member of ERM protein family, bridging cytoskeleton and plasma membrane in a variety of cells, participating in cell morphology maintenance and cell adhesion. Several cell functions such as cell migration and signal transduction. Some studies have shown that the family proteins are involved in the regulation of mouse oocyte cortical tension and the localization of the spindle during the second maturation and division. The active forms of Ezrin and phosphorylation were present in mouse oocytes from GV phase to MII stage, but it was not clear what role they played in the maturation process of mouse oocytes. The role of the protein in mouse oocyte maturation and its effect on embryo development potential after fertilization were studied by microinjection of specific siRNA knockdown Ezrin. The contents and results of the study are as follows: 1. After microinjection of Ezrin-siRNA, the effect of the protein on the development and maturation of mouse oocytes was studied. At the same time, the expression level of Ezrin in the nude eggs of mice at GV stage was microinjected with Ezrin-siRNA or NC-siRNA (negative control group) into the nude eggs of GV phase, and was inhibited in Milrinone- containing (inhibiting nuclear mitotic M 2) for 0 h, 6 h, 12 h, 18 h, 18 h, 24 h, 30 h and 36 h, respectively. Ezrin specific antibody was used for fluorescence staining and Image J software was used to analyze the average fluorescence value of the images. The results showed that: during 1830 h after microinjection, The expression of Ezrin in the experimental group was significantly lower than that in the control group (P 0.05). 2. The effect of Milrinone on the maturation rate of mouse GV naked eggs at different time. The mice GV naked eggs were blocked in Milrinone medium for 0 h (control group 6 h, 12 h, 18 h, 18 h, 24 h) for 30 h and 36 h, respectively. The maturation rate of oocytes in each group was calculated by excretion of PBI. When the block time was less than 18 h, the maturation rate of oocytes in each group was not significantly different from that in the control group (P0.05), but the maturation rate of oocyte was significantly lower than that of the control group and the control group (鈮，

本文編號(hào)：1549838