冷冻保存时长对卵裂期胚胎组蛋白表观遗传修饰的影响

Effect of Long-time Slow-frozen Storage on Epigenetic Changes of Histone in Cleavage-stage Embryos

【目的】研究不同的冷冻保存时长对人类卵裂期胚胎的组蛋白表观遗传修饰的影响。【方法】采用经患者知情同意捐献用于科研的慢速冷冻的卵裂期胚胎,根据冷冻时长分为6、9和12年组,以新鲜胚胎为对照组,每组6枚捐献胚胎。采用细胞免疫荧光技术观察去乙酰化酶HDAC1、组蛋白(H3K9ac、H3K4me3、H3K9me3)的表达量在各组胚胎中的差异,并以单细胞RT-qPCR技术检测去乙酰化酶HDAC1、甲基化酶(SUV39H1、SET?DB1)、去甲基化酶KDM5A对应mRNA的表达量。【结果】4组胚胎的去乙酰化酶HDAC1、组蛋白(H3K9ac、H3K4me3和H3K9me3)的蛋白表达量无统计学意义的差异(P>0.05)。同时去乙酰化酶HDAC1、甲基化酶SUV39H1对应mRNA的表达量也无统计学意义的差异(P>0.05)。随着胚胎冷冻时长的增加,甲基化酶SETDB1对应mRNA的表达量有递增趋势,然而差异无统计学意义(P>0.05)。去甲基化酶KDM5A对应的mRNA的表达量也随冷冻时长的增加而增强,差异有统计学意义(P<0.05)。【结论】慢速冷冻保存时长对卵裂期胚胎的组蛋白(H3K9ac、H3K4me3和H3K9me3)的蛋白表达量无显著影响,甲基化酶(SUV39H1、SETDB1)对应的mRNA表达量没有明显影响;但随着冷冻保存时长增加,胚胎去甲基化酶KDM5A对应的mRNA表达量增加,有可能导致胚胎转录抑制程度增强。

【Objective】To investigate the impact of long-term storage time on epigenetic modification of histone in human cleavage stage embryos.【Methods】According to the length of storage time,donated embryos after slow-freezing were divided into 3 groups:6-year group,9-year group,and 12 -year group,while the control group consisted of donated fresh embryos. Immunocytochemistry was performed to compare the expression levels of HDAC1, H3K9ac, H3K4me3,and H3K9me3 among 4 groups. Transcription levels of HDAC1,SUV39H1,SETDB1,and KDM5A were analyzed through Single-Cell qRT-PCR.【Results】The relative abundances of HDAC1 and SUV39H1 mRNA showed no significant differences among 4 groups(P > 0.05). SETDB1 exhibited a climbing pattern as storage time increased,but no significant difference was observed(P > 0.05). There were no differences in H3K9 trimethylation and H3K9 methylation among 4 groups. However,the expression level of KDM5A increased with the increasing storage time(P < 0.05).【Conclusions】Storage time did not affect the expression of deacetylase HDAC1,methylase SUV39H1 and SETDB1. H3K9ac/me3 and H3K4me3 also exhibited no significant difference as the storage time increases. However,the increasing storage length might induce the elevating expression of demethylase KDM5A,which may be associated with inhibition of embryonic transcription.