姜黄素对黄牛卵母细胞体外成熟质量的影响

发布时间：2018-06-17 14:45

  本文选题：姜黄素 + 卵母细胞　； 参考：《广西大学》2017年硕士论文

【摘要】：本研究以黄牛卵母细胞为研究对象,通过探讨姜黄素(Curcumin)对黄牛卵母细胞体外成熟(Matured in vitro,IVM)及其随后体外受精胚胎发育的影响,并结合卵母细胞内线粒体合成基因的表达及线粒体生物活性的变化情况,探究姜黄素对黄牛卵母细胞体外成熟的影响及其作用机制,为进一步优化黄牛卵母细胞体外成熟培养体系提供理论依据。首先,探讨了姜黄素对黄牛卵母细胞体外成熟质量及卵母细胞早期胚胎发育的影响。分别添加不同浓度的姜黄素(Oμmol/L、5μmol/L、10μmol/L、15μmol/L)到卵母细胞成熟液中用以培养黄牛卵丘-卵母细胞复合体(COCs)24h。结果显示:添加1Oμmol/L姜黄素处理组的卵母细胞成熟率显著高于5μμmol/L、15μmol/L 处理组和对照组(73.00%±4.07%vs 63.29%±4.76%,65.23%±2.73%,62.04%±2.98%,p0.05)。同时对成熟之后的黄牛卵母细胞进行体外受精的研究发现:成熟液中添加1Oμmol/L姜黄素与对照组相比能显著提高黄牛卵母细胞体外受精后胚胎卵裂率(78.16%±2.19%vs 70.18%±2.95%)及囊胚发育率(28.19%±2.91%vs21.58%±3.54%,p0.05);且对囊胚细胞数的检测发现,10μmol/L姜黄素处理组囊胚的细胞数要显著高于对照组(110.2±7.48vs103.43±7.04,p0.05)。其次,探讨了姜黄素提高黄牛卵母细胞体外成熟率的作用机制。通过荧光定量PCR对体外成熟培养后黄牛卵母细胞线粒体转录相关基因进行检测,结果发现:姜黄素各处理组的MⅡ期黄牛卵母细胞内线粒体生物合成的中枢调节因子PGC-1α、及线粒体转录因子A(TFAM)和解偶联蛋白2(UCP2)的mRNA表达水平与对照组相比均有着显著地上调(p0.05)。采用Hochest染色法检测黄牛卵母细胞质内TFAM蛋白表达的分析结果显示:10μmol/L姜黄素处理组的MⅡ期黄牛卵母细胞TFAM蛋白的表达水平明显地高于对照组(p0.05)。利用线粒体荧光探针对MⅡ期黄牛卵母细胞进行染色观察发现,1Oμmol/L姜黄素处理组和对照组的MⅡ期黄牛卵母细胞内线粒体在整个卵子胞质内呈均匀分布,但1Oμmol/L姜黄素处理组的线粒体相对荧光强度要显著高于对照组(p0.05)。同时对黄牛卵母细胞体外成熟培养后细胞内ATP的检测结果显示:1Oμmol/L姜黄素处理组中MⅡ期卵母细胞内的ATP含量略微低于对照组,但差异不显著(P0.05)。线粒体DNA拷贝数相对表达量分析发现:1Oμmol/L姜黄素的处理组卵母细胞mtDNA拷贝数显著高于对照组(p0.05)。以上结果表明:(1)黄牛卵母细胞体外成熟培养液中添加适宜浓度(1Oμmol/L)的姜黄素有利于促进黄牛卵母细胞体外成熟及其随后胚胎的早期发育;(2)姜黄素通过促进卵母细胞中线粒体生物合成中枢基因PGC-1α和转录因子TFAM及解偶联蛋白UCP2表达的上调,促进线粒体生物合成,从而提高内源性线粒体含量及活性进而提高黄牛卵母细胞体外成熟质量。
[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% 卤4.07%vs 63.29% 卤4.76% 卤2.73% 卤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% 卤2.19%vs 70.18% 卤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 卤7.48vs103.43 卤7.04 7.48vs103.43 卤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% 卤2.91% 卤3.54% 卤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 伪 and mitochondrial transcription factor AtFAM and conjugate protein 2 (UCP2) in M 鈪，

本文编号：2031407