本文選題：姜黃素 + 卵母細胞��； 參考：《廣西大學》2017年碩士論文

【摘要】：本研究以黃牛卵母細胞為研究對象,通過探討姜黃素(Curcumin)對黃牛卵母細胞體外成熟(Matured in vitro,IVM)及其隨后體外受精胚胎發(fā)育的影響,并結合卵母細胞內線粒體合成基因的表達及線粒體生物活性的變化情況,探究姜黃素對黃牛卵母細胞體外成熟的影響及其作用機制,為進一步優(yōu)化黃牛卵母細胞體外成熟培養(yǎng)體系提供理論依據(jù)。首先,探討了姜黃素對黃牛卵母細胞體外成熟質量及卵母細胞早期胚胎發(fā)育的影響。分別添加不同濃度的姜黃素(Oμmol/L、5μmol/L、10μmol/L、15μmol/L)到卵母細胞成熟液中用以培養(yǎng)黃牛卵丘-卵母細胞復合體(COCs)24h。結果顯示:添加1Oμmol/L姜黃素處理組的卵母細胞成熟率顯著高于5μμmol/L、15μmol/L 處理組和對照組(73.00%±4.07%vs 63.29%±4.76%,65.23%±2.73%,62.04%±2.98%,p0.05)。同時對成熟之后的黃牛卵母細胞進行體外受精的研究發(fā)現(xiàn):成熟液中添加1Oμmol/L姜黃素與對照組相比能顯著提高黃牛卵母細胞體外受精后胚胎卵裂率(78.16%±2.19%vs 70.18%±2.95%)及囊胚發(fā)育率(28.19%±2.91%vs21.58%±3.54%,p0.05);且對囊胚細胞數(shù)的檢測發(fā)現(xiàn),10μmol/L姜黃素處理組囊胚的細胞數(shù)要顯著高于對照組(110.2±7.48vs103.43±7.04,p0.05)。其次,探討了姜黃素提高黃牛卵母細胞體外成熟率的作用機制。通過熒光定量PCR對體外成熟培養(yǎng)后黃牛卵母細胞線粒體轉錄相關基因進行檢測,結果發(fā)現(xiàn):姜黃素各處理組的MⅡ期黃牛卵母細胞內線粒體生物合成的中樞調節(jié)因子PGC-1α、及線粒體轉錄因子A(TFAM)和解偶聯(lián)蛋白2(UCP2)的mRNA表達水平與對照組相比均有著顯著地上調(p0.05)。采用Hochest染色法檢測黃牛卵母細胞質內TFAM蛋白表達的分析結果顯示:10μmol/L姜黃素處理組的MⅡ期黃牛卵母細胞TFAM蛋白的表達水平明顯地高于對照組(p0.05)。利用線粒體熒光探針對MⅡ期黃牛卵母細胞進行染色觀察發(fā)現(xiàn),1Oμmol/L姜黃素處理組和對照組的MⅡ期黃牛卵母細胞內線粒體在整個卵子胞質內呈均勻分布,但1Oμmol/L姜黃素處理組的線粒體相對熒光強度要顯著高于對照組(p0.05)。同時對黃牛卵母細胞體外成熟培養(yǎng)后細胞內ATP的檢測結果顯示:1Oμmol/L姜黃素處理組中MⅡ期卵母細胞內的ATP含量略微低于對照組,但差異不顯著(P0.05)。線粒體DNA拷貝數(shù)相對表達量分析發(fā)現(xiàn):1Oμmol/L姜黃素的處理組卵母細胞mtDNA拷貝數(shù)顯著高于對照組(p0.05)。以上結果表明:(1)黃牛卵母細胞體外成熟培養(yǎng)液中添加適宜濃度(1Oμmol/L)的姜黃素有利于促進黃牛卵母細胞體外成熟及其隨后胚胎的早期發(fā)育;(2)姜黃素通過促進卵母細胞中線粒體生物合成中樞基因PGC-1α和轉錄因子TFAM及解偶聯(lián)蛋白UCP2表達的上調,促進線粒體生物合成,從而提高內源性線粒體含量及活性進而提高黃牛卵母細胞體外成熟質量。
[Abstract]:In this study, the effect of curcumin on matured oocytes matured in vitro (IVM) and subsequent embryo development in vitro fertilization (IVM) was studied. The effect of curcumin on the maturation of bovine oocytes in vitro and its mechanism were investigated by combining the expression of mitochondrial synthetic genes and the changes of mitochondrial biological activities in oocytes. It provides theoretical basis for further optimization of maturation system of bovine oocytes in vitro. Firstly, the effect of curcumin on maturation quality and early embryo development of bovine oocytes was studied. Different concentrations of curcumin O 渭 mol 路L ~ (-1) 5 渭 mol 路L ~ (-1) 10 渭 mol / L ~ (10) 渭 mol / L ~ (15 渭 mol / L) were added to the oocyte maturation fluid to culture bovine cumulus / oocyte complex (COCsN) for 24 h. The results showed that the maturation rate of oocytes in curcumin treated with 10 渭 mol / L curcumin was significantly higher than that in 5 渭 mol / L 15 渭 mol / L and control group (73.00% 鹵4.07%vs 63.29% 鹵4.76% 鹵2.73% 鹵2.73%). The results of in vitro fertilization of bovine oocytes after maturation showed that addition of 1 0 渭 mol / L curcumin to maturation fluid significantly increased cleavage rate of embryos after in vitro fertilization of bovine oocytes and blastocyst development (78.16% 鹵2.19%vs 70.18% 鹵2.95%) and blastocyst development. The number of blastocyst cells in the 10 渭 mol / L curcumin treated group was significantly higher than that in the control group (110.2 鹵7.48vs103.43 鹵7.04 7.48vs103.43 鹵7.04 p 0.05), and the number of blastocyst cells in the 10 渭 mol / L curcumin treated group was significantly higher than that in the control group (28.19% 鹵2.91% 鹵3.54% 鹵3.54%). Secondly, the mechanism of curcumin increasing maturation rate of bovine oocytes in vitro was discussed. Mitochondrial transcription related genes in cultured bovine oocytes were detected by fluorescence quantitative PCR. The results showed that the mRNA expression levels of central regulatory factor PGC-1 偽 and mitochondrial transcription factor AtFAM and conjugate protein 2 (UCP2) in M 鈪，

本文編號：2031407