孕妇外周血中RASSF1A基因甲基化状态研究

Analysis of RASSF1A gene methylation status in maternal plasma

目的探讨RASSF1A基因作为孕妇外周血中胎儿特异性标记物的可行性。方法随机选取43例的外周血样本,其中妊娠晚期孕妇标本40例、健康未妊娠女性标本3例,以及早期妊娠行流产的绒毛组织样本5例。利用甲基化敏感限制性内切酶PCR(MSRE-PCR)及荧光定量PCR的方法,检测血细胞、绒毛和血浆DNA的RASSF1A基因甲基化状态。结果健康未妊娠妇女血浆及孕妇血细胞中均未检出甲基化RASSF1A基因;绒毛组织中甲基化RASSF1A基因检出率为100%;孕妇血浆中甲基化RASSF1A基因检出率为92.5%。妊娠晚期孕妇血浆RASSF1A基因含量Ct平均值为30.43±2.37。结论在母体和胎儿DNA中,RASSF1A基因甲基化状态有明显差异,用实时荧光定量PCR可对孕妇血浆RASSF1A基因进行定量,提示了RASSF1A基因是无创产前诊断的一个潜在标记物。

Objection：Explore the feasibility of RASSF1A gene as the fetal specific marker of pregnant women in peripheral blood. Methods：43 cases of peripheral blood samples were randomly selected,including the third trimester pregnant women 40 cases,non-pregnant women specimen 3 cases,and 5 cases of chorionic tissue samples in early pregnancy abortion. DNA were extracted from peripheral blood leukocytes,chorionic villus and plasma,then RASSF1A methylation status were analyzed using methylation sensitive restriction enzyme PCR（MSRE-PCR）and real time PCR. Results：Methylated RASSF1A gene was not detected from the DNA of non-pregnant women plasma and pregnant women peripheral blood leukocytes;Detection rate of methylated RASSF1A gene in villi tissue was 100% and 92.5% in maternal plasma.The mean Ct values of methylated RASSF1A gene in the third trimester pregnancy groups was 30.43±2.37. Conclusion：Methylation status of RASSF1A gene in maternal DNA and fetal DNA were different. Concentration of methylated RASSF1A gene in maternal plasma can be measured by real time PCR,which show that methylated RASSF1A gene can be a potential marker of non-invasive prenatal diagnostic methods.