甲基化DNA免疫沉淀-荧光实时定量PCR检测人精浆游离DNA睾丸和附睾特异性甲基化启动子  

Detecting testis-and epididymis-specific methylated promoters in human cell-free seminal DNA by MeDIP-qPCR

作　　者：官黄涛[1,2] 吕金明 吴春林[1,4] 李红钢[1,2] 朱长虹[1,2] 熊承良[1,2] 

机构地区：[1]华中科技大学同济医学院计划生育研究所/生殖医学中心,湖北武汉430030 [2]武汉同济生殖医学专科医院,湖北武汉430030 [3]蕲春县计划生育服务站,湖北黄冈435300 [4]武汉市第一医院生殖医学中心,湖北武汉430022

出　　处：《中华男科学杂志》2013年第11期977-983,共7页National Journal of Andrology

基　　金：国家科技"十二五"计划项目(No.2012BAI32B03);国家教育部博士点基金(No.20090142110034)~~

摘　　要：目的:建立检测精浆游离DNA(cfsDNA)启动子甲基化水平的甲基化DNA免疫沉淀-荧光实时定量PCR(MeDIP-qPCR)方法。方法:提取精液参数正常(6例)和输精管结扎(6例)男性的cfsDNA,将两组不同个体的cfsDNA分别进行混合,超声处理后形成DNA片段,通过MeDIP分离甲基化DNA片段,然后用qPCR方法检测启动子甲基化水平。结果:精液参数正常组的PRAME、PEG10、MORC1、GML、HOXA5、DNMT3L、SNURF、MSH4、DAZ1和CLPB启动子甲基化水平分别为14.93%、2.64%、0.69%、2.66%、17.50%、21.10%、5.98%、2.28%、13.50%和3.86%,明显低于输精管结扎组的121.25%、73.62%、16.25%、42.90%、76.74%、112.40%、59.79%、25.85%、91.90%和64.53%。其中,PRAME、MORC1、GML、HOXA5、DNMT3L、SNURF、MSH4和DAZ1等8个启动子MeDIP-qPCR的检测结果与启动子甲基化芯片结果一致。结论:通过MeDIP-qPCR方法可以定量检测到cfsDNA中的特异性启动子甲基化,为确定cfsDNA中睾丸和附睾特异性甲基化启动子提供了有效途径。[ Abstract] Objective： To establish a method of methyl-DNA immunoprecipitation （MeDIP）-real time quantitative PCR （qPCR） for detecting the promoter methylation level in cell-free seminal DNA （cfsDNA）. Methods： We obtained cfsDNA samples from 6 normozoospermia men （the NZ group） and 6 post-vasectomy patients （the PV group）, and mixed the samples from different in- dividuals of each group, respectively. Then we made DNA fragments by uhrasonication, separated the methylated DNA fragments by MeDIP, and determined the methylation level of the promoters in cfsDNA by qPCR. Results ： The methylation levels of the promoters PRAME, PEG10, MORC1, GML, HOXA5, DNMT3L, SNURF, MSH4, DAZ1 and CLPB were 14.93, 2.64, 0.69, 2.66, 17.50, 21.10, 5.98, 2.28, 13.50 and 3.86% , respectively, in the NZ group, obviously lower than 121.25, 73.62, 16.25, 42.90, 76.74, 112.40, 59.79, 25.85, 91.90 and 64.53% in the PV group. The results of MeDIP-qPCR for the methylation of PRAME, MORC1, GML, HOXA5, DNMT3L, SNURF, MSH4 and DAZ1 were coincident with the results of genome-wide promoter methylation microarray. Conclusion： MeDIP-qPCR can quantitatively measure the promoter methylation level in cfsDNA, and effectively deter- mine the testis- and epididymis-specific methylated promoters in human semen. Natl J Androl, 2013, 19 （11） ： 977-983

关 键 词：精浆游离DNA 甲基化DNA免疫沉淀-荧光实时定量PCR 输精管结扎 睾丸 附睾 启动子 

分 类 号：R446.6[医药卫生—诊断学]