摘要
目的通过体外分离成熟的精子,利用实时荧光定量PCR(qRealTime-PCR)检测其是否表达DNA从头甲基化转移酶(DNMT1/3a/3b)及甲基CpG结合蛋白(MeCP2),为精子中甲基化表观修饰研究提供依据。方法体外采集健康男性志愿者的精液标本,利用精子上游收集法获得具有活性的成熟精子。通过SYBR Green/PI双染色法,利用流式细胞术(FCM)检测精子样本的质膜完好性及其活性。抽提精子的总RNA,逆转录PCR获得cDNA,利用qRealTime-PCR鉴定精子甲基化转移酶及甲基CpG结合蛋白的mRNA表达情况。结果SYBR Green/PI双染色和流式细胞仪检测发现,利用上游收集法可以收集SYBR Green+/PI-的精子占总精子数的(94.513±1.120)%,表明该方法可以收集到质膜完好且活性强的精子。qRealTime-PCR结果显示,此样本精子中DNA从头甲基化转移酶的表达比较明显,而甲基CpG结合蛋白的表达量相当低。其中DNMT1的mRNA表达量较高(0.272±0.045,P<0.05),DNMT3a和DNMT3b的表达水平比较弱[(4.930±0.01)E-006,(6.500±1.7)E-005,P<0.05],而MeCP2几乎不表达([2.12±0.91)E-006,P<0.05]。结论利用qRealTime-PCR可以比较快速方便的检测出此次精子样本中DNA从头甲基化转移酶和甲基CpG结合蛋白的mRNA表达水平。
Objective To investigate the mRNA expression of de novo DNA Methyltransferase （DNMT1/3a/3b） and methyl-CpG Binding Protein2 （MeCP2） in spermatozoon by qRealTime-PCR. Methods Human ejaculates were obtained from healthy volunteers.Activated mature sperms were purified and collected by swim-up method. The integrality and activity of spermatozoon was detected by flow cytometry with SYBR Green/PI double-staining. The expression of de novo DNA Methyltransferase and methyl-CpG Binding Protein2 in spermatozoon were also examined by RT-PCR and qRealTime-PCR. Results Activated mature spermatozoon with SYBR Green＋/PI＋ accounted for （94.513±1.120）%. The results of qRealtime- PCR indicated that the mRNA expression of de novo DNA Methyltransferase in this samples were remarkablely strong,but the mRNA of methyl-CpG Binding Protein2 was poor.The mRNA of D .NMT1 expressed is higher（0.272±0.045, P〈0.05） than that of DNMT3a＇s and DNMT3b＇s [（4.930±0.01）E-006,（6.500±1.7）E-005, P〈0.05]. However, almost no mRNA expression of MeCP2 were detected[（2.12±0.91）E-006, P〈 0.05]. Conclusion The mRNAs of de novo DNA Methyltransferase and methyl- CpG Binding Protein2 were detected by qRealTime-PCR in spermatozoon.
出处
《中国男科学杂志》
CAS
CSCD
2008年第8期5-9,15,共6页
Chinese Journal of Andrology
基金
国家自然科学基金资助(NSFCNo.30470857)
