玻璃化冷冻对小鼠囊胚线粒体DNA及线粒体相关基因表达影响研究

摘要

目的 探讨玻璃化冷冻对小鼠囊胚线粒体DNA(mtDNA)和线粒体相关基因mRNA表达的影响.方法 本研究于2015年6月至2016年1月中山大学北校区实验动物中心完成.将180个小鼠囊胚分成玻璃化冷冻组(n=90)和新鲜组(n=90),每组30个胚胎用于mtDNAD环(D-loop)区测序分析;30个胚胎用于实时荧光定量PCR测mtDNA拷贝数;剩余胚胎用于实时荧光定量PCR检测线粒体转录因子A(Tfam)和DNA聚合酶γ(Polg1和Polg2)的mRNA表达,比较2组碱基突变率和mtDNA拷贝数及线粒体相关基因mRNA表达.结果 对照组和玻璃化冷冻组均未发现碱基突变;对照组和玻璃化冷冻组的胚胎mtDNA平均拷贝数分别为2.9×10(s=1.4×105)和3.2×105(s=1.8×105),差异无统计学意义(P＞0.05);两组胚胎的3个线粒体相关基因mRNA表达差异均有统计学意义(P＜0.05),玻璃化冷冻组的3个基因mRNA表达均上调.结论 玻璃化冷冻对小鼠囊胚mtDNA没有影响,但是会引起线粒体相关基因mRNA的表达上调.%Objective This study aimed to investigate the effect of vitrification on mitochondrial DNA (mtDNA) and the mRNA expression of mitochondria-related genes in mouse blastocysts.Methods The experiment was performed in the Experimental Animal Center of the North Campus of Sun Yat-Sen University from June 2015 to January 2016.180 mouse blastocysts were divided into vitrification group (n=90)and fresh group (n=90),30 blastocysts in vitrification group and 30 blastocysts in fresh group were used for sequencing to analyse mtDNA D-loop region;and 30 blastocysts in each group were used for quantitative real-time PCR to detect mtDNA copy number;the remaining embryos were used for detecting the mRNA expression of mitochondria-related genes,including mitochondrial transcription factor A (Tfam) and DNA polymerase γ (Polg1 and Polg2),then we compared the base mutation rate,mtDNA copy number and the mRNA expression of mitochondria-related genes between two groups.Results There was no mutation in both groups;the mean mtDNA copy number was 2.9× 105(s=1.4× 105) and 3.2× 105(s=1.8× 105) in fresh group and vitrification group,respectively,but there was no significant differences (P ＞ 0.05);the mRNA expression of mitochondria-related genes was statistically different between groups(P ＜ 0.05),the expression level of three genes in vitrification group increased.Conclusion Vitrification has no effect on mtDNA in mouse blastocysts,but it can cause increased mRNA expression of mitochondria-related genes.