少弱畸形精子症患者H19及MEST基因DNA甲基化分析  

Aberrant DNA methylation of H19 and MEST in oligoastheno-teratozoospermias male sperm

作　　者：张伟娜[1,2] 刘志伟[2] 上官陈燕[2] 陆军[2] 刘睿智[1] 于洋[1] 

机构地区：[1]吉林大学第一医院生殖医学中心产前诊断中心,长春130021 [2]东北师范大学遗传与细胞研究所

出　　处：《中华泌尿外科杂志》2013年第6期422-425,共4页Chinese Journal of Urology

基　　金：2011年国家人口和计划生育委员会科学技术司项目（2011-GJKJS4）7）

摘　　要：目的比较正常生育男性及少弱畸形精子症患者父系印记基因H19及母系印记基因MESTDNA甲基化的差异。方法对2011年3月至2012年2月15例正常生育男性和33例少弱畸形精子症患者精液标本进行常规分析和精子形态分析。密度梯度离心法提纯精液，提取精液基因组DNA并进行亚硫酸氢盐处理，利用PCR体外扩增并将纯化后的PCR产物与pMD R 18-T载体连接及酶切验证，并对阳性克隆进行测序和DNA甲基化程度差异分析。结果正常生育男性H19DNA高甲基化5例，甲基化率为100．0％，10例少弱畸形精子症患者DNA甲基化水平显著下降，甲基化率为93．0％，两组比较差异有统计学意义（P〈0．01）。正常生育男性MESTDNA低甲基化10例，甲基化率为1．6％，23例少弱畸形精子症患者DNA甲基化率为4．8％，两组比较差异无统计学意义（P〉0．05）。结论H19差异甲基化区域的甲基化异常和男性不育有关，可能成为人类精子形成缺陷的生物学标志之一。Objective To study the aberrant DNA methylation of H19 and MEST in infertile male sperm. Methods Routine semen analysis and morphological analysis were performed on 15 control indi-viduals and 33 oligoastheno-teratozoospermias. Sperms were purified by density gradient centrifugation and DNA was extracted and treated with bisulfite. After PCR, the purified PCR products were cloned into pMD R 18-T vector and proceed to chemical transformation, restriction enzyme reaction. For each sample, posi-tive clones were selected for sequencing analysis and DNA methylation analysis. Results Five control in-dividuals had high degree of H19 and methylation rate was 100.0% , while the methylation rate of 93.0% in 10 oligoastheno-teratozoospermias was significantly lower. Significant difference was found in methylation rate between the control individuals and oligoastheno-teratozoospermias （ P 〈 0.01 ）. As to MEST, 10 control in- dividuals had a low degree of MEST and the methylation rate was 1.6% , while the methylation rate in 23 oli-goastheno-teratozoospermias was 4.8%. No significant difference was found in the methylation rate between control individuals and oligoastheno-teratozoospermias （P 〉 0.05）. Conclusion Male infertility is associ-ated with aberrant mcthylation of H19 differentially methylated region, and it may be a biomarker of human spermatogenesis defeet.

关 键 词：男性不育 精子发生 基因转变 甲基化印记 

分 类 号：R698.2[医药卫生—泌尿科学]