摘要目的 通过对体外培养获取的昆明白小鼠单倍体精子细胞印迹基因H19印迹控制区(imprinting control region,ICR)和IGF2r差异甲基化区(differentially methylated region,DMR2)甲基化状态的检测,初步评估体外培养对生精细胞印迹基因甲基化状态的影响.方法 应用亚硫酸氢盐测序技术,以印迹基因H19 ICR和IGF2r DMR2区为扩增目的区域,对经体外培养获得的单倍体精子细胞进行甲基化修饰后巢式和半巢式PCR扩增,应用DNAman软件对特异性扩增产物测序分析,与GenBank相对应的序列比对,判断其甲基化状态,并与小鼠附睾内成熟精子进行比较.结果 小鼠成熟精子中H19 ICR区15个CpG位点呈高度甲基化状态(96.67％),IGF2r DMR2区呈非甲基化状态(94.29％);而经体外培养获得的单倍体精子细胞H19 ICR区甲基化程度显著降低(69.33％,P＜0.01),IGF2r DMR2区甲基化程度显著增加(44.29％,P＜0.01).结论 昆明白小鼠成熟精子中H19 ICR区呈高度甲基化状态;IGF2r DMR2区呈非甲基化状态;体外培养产生的单倍体精子细胞H19 ICR区出现了广泛的甲基化丢失,IGF2r DMR2区出现了异常甲基化.

abstractsObjective To compare the methylation patterns of imprinting control region (ICR) of H19 gene and differentially methylated region (DMR) of IGF2r gene in mature sperms derived from epididymis of Kunming mice and in vitro cultured haploid spermatids.Methods The H19 ICR and IGF2r DMR2 were detected by bisulfite sequencing PCR (BSP).The results were compared with standard sequence derived from GenBank using a DNAman software.Results 96.67％ (15 CpG sites) of H19 ICR was found to be methylated,and 94.29％ IGF2r DMR2 was found to be unmethylated in mature sperms.By contrast,69.33％ of H19 ICR and 44.29％ of IGF2r DMR2 were found to be methylated in the haploid spermatids cultured in vitro.A significant difference was detected in the methylation patterns between the two types of cells (P＜0.01).Conclusion The H19 ICR in mature sperm of Kunming mice was essentially methylated,while the IGF2r DMR2 was essentially unmethylated.Partial methylation loss in H19 ICR and abnormal methylation in IGF2r DMR2 were found in the haploid spermatids cultured in vitro.