小鼠生精细胞体外培养过程中印迹基因H19和IGF2r甲基化状态  

Methylation pattern of imprinted genes H19 and IGF2r in spermatogenic cells cultured in vitro

作　　者：袁韦娜[1,2] 姜宏[3] 张文香[3] 汪存利[3] 

机构地区：[1]安徽医科大学解放军临床学院生殖医学中心,合肥230031 [2]合肥市第一人民医院妇产科 [3]解放军第105医院生殖医学中心

出　　处：《中华医学遗传学杂志》2013年第6期678-682,共5页Chinese Journal of Medical Genetics

基　　金：安徽省自然科学基金（090413271x）

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（imprintingcontrolregion，ICR）和IGF2r差异甲基化区（differentiallymethylatedregion，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H19ICR和IGF2rDMR2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H19ICR区15个CpG位点呈高度甲基化状态（96．67％），IGF2rDMR2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H19ICR区甲基化程度显著降低（69．33％，P〈0．01），／GF2rDMR2区甲基化程度显著增加（44．29％，P〈O．01）。结论昆明白小鼠成熟精子中H19ICR区呈高度甲基化状态；IGF2rDMR2区呈非甲基化状态；体外培养产生的单倍体精子细胞H19ICR区出现了广泛的甲基化丢失，IGF2rDMR2区出现了异常甲基化。Objective 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.

关 键 词：生精细胞 体外培养 印迹基因 DNA甲基化 

分 类 号：R714.8[医药卫生—妇产科学]