白藜芦醇和褪黑素对绵羊胎儿成纤维细胞转染及卵母细胞体外成熟的影响

试验旨在利用白藜芦醇(Re)和褪黑素(MT)提高绵羊胎儿成纤维细胞电穿孔转染(电转)效率、优化卵母细胞体外成熟体系,以提高转基因动物生产效率。将绵羊胎儿成纤维细胞分为6组:对照组、添加白藜芦醇高、中、低(R^-5、R^-6、R^-7)3种浓度组及白藜芦醇和褪黑素联合添加组(R^-5M^-5、R^-6M^-7),通过电转效率和细胞凋亡情况确定电转体系优化方案;通过对细胞周期、线粒体膜电位及活性氧(ROS)的检测研究其作用机制。将绵羊卵母细胞分为对照组及白藜芦醇和褪黑素(R^-5M^-5、R^-6M^-7)联合添加组,通过绵羊卵母细胞体外成熟后卵丘细胞扩展、第一极体排出及体外受精胚胎的卵裂率检测其对卵母细胞体外成熟的作用。研究结果显示,分离的绵羊胎儿成纤维细胞P3代增殖旺盛,细胞生长曲线呈典型的S型;与对照组相比,所有试验组胎儿成纤维细胞的电转效率均显著提高(P<0.05),R^-5M^-5组电转效率最高;R^-6M^-7组显著降低细胞凋亡率(P<0.05),R^-5、R^-6、R^-5M^-5和R^-6M^-7组均显著促进细胞增殖(P<0.05);R^-6、R^-7、R^-5M^-5和R^-6M^-7组G1期细胞增加,S期细胞相对减少,但差异均不显著(P>0.05);R^-6、R^-7、R^-5M^-5和R^-6M^-7组成纤维细胞内ROS水平均显著降低(P<0.05),R^-6、R^-5M^-5和R^-6M^-7组线粒体膜电位均显著增加(P<0.05);R^-6M^-7组显著促进绵羊卵丘细胞扩展、提高卵母细胞第一极体排出率和体外受精胚胎发育能力(P<0.05)。综上,白藜芦醇和褪黑素可以优化绵羊胎儿成纤维细胞电转体系,提高卵母细胞体外成熟效率,为白藜芦醇和褪黑素在转基因动物生产中的应用提供参考依据。

Effects of Resveratrol and Melatonin on Transfection of Sheep Fetal Fibroblasts and in vitro Maturation of Oocytes

This experiment was aimed to use resveratrol (Re) and melatonin (MT) to improve the efficiency of electroporation transfection of sheep fetal fibroblasts, and optimize the in vitro maturation system of oocytes, in order to improve the production efficiency of transgenic animals.Sheep fetal fibroblasts were divided into control group, high, medium and low concentrations of resveratrol groups (R^-5, R^-6 and R^-7), resveratrol and melatonin combined groups (R^-5M^-5and R^-6M^-7), 6 groups in total.The optimization scheme of electrotransfer system was determined by electrotransfection efficiency and cell apoptosis.The mechanism was studied by detecting cell cycle, mitochondrial membrane potential and reactive oxygen species (ROS).Sheep oocytes were divided into control group and combined addition group of resveratrol and melatonin (R^-5M^-5, R^-6M^-7), the effect of ovine oocytes on in vitro maturation was detected by cumulus cell expansion, first polar body excretion and cleavage rate of in vitro fertilized embryos.The results showed that the P3 generation of isolated sheep fetal fibroblasts proliferated vigorously, and the cell growth curve showed a typical S shape.When compared with control group, the electrotransfection efficiency of sheep fetal fibroblasts in all experimental groups was significantly increased (P<0.05), R^-5M^-5 group had the highest electrotransfection efficiency, R^-6M^-7 group significantly decreased the cell apoptosis rate (P<0.05), R^-5, R^-6, R^-5M^-5 and R^-6M^-7 groups significantly promoted the cell proliferation (P<0.05), R^-6, R^-7, R^-5M^-5 and R^-6M^-7 groups increased the proportion of G1 phase cells, while S phase cells decreased relatively, but the difference was not significant (P>0.05).ROS level was significantly decreased in R^-6, R^-7, R^-5M^-5 and R^-6M^-7 groups (P<0.05), and mitochondrial membrane potential was significantly increased in R^-6, R^-5M^-5 and R^-6M^-7 groups (P<0.05).R^-6M^-7 group significantly promoted the expansion of cumulus cells, and significantly increased the rate of first polar body extrusion and the development ability of in vitro fertilization embryo (P<0.05).In conclusion, resveratrol and melatonin could optimize the electroporation transfection system, improve the in vitro maturation efficiency of oocytes, and provide references for the application of resveratrol and melatonin in the production of transgenic animals.