母血中胎源性RASSF1A基因的检测及其在子痫前期中的临床意义

Quantitative Analysis of Placenta-derived Fetal Specific RASSF1A Sequences in Maternal Blood and Its Clinical Significance in Pre-eclamptic Pregnancies

目的:初步探讨孕妇血浆中胎源性Ras相关区域家族1A(Ras association domain family 1A,RASSF1A)基因的动力学特点及其在子痫前期中的含量变化。方法:应用2种甲基化敏感的限制性内切酶HinP1I和HhaI去除孕妇血浆中非甲基化的RASSF1A序列,保留超甲基化RASSF1A序列。通过含目的基因的质粒标准品绘制标准曲线,结合荧光定量PCR(FQ-PCR)技术测定血浆中酶切后RASSF1A基因的含量,反映游离胎儿DNA水平。同时检测β-actin基因确保酶对非甲基化序列的完全消化。结果:①最早可在妊娠7+3周检测出孕妇血浆中超甲基化的RASSF1A基因;②孕妇血浆中超甲基化RASSF1A基因的含量随妊娠的进程逐渐增加,且在分娩后24 h内消失;③未妊娠组中均未检测到超甲基化的RASSF1A基因;④妊娠晚期子痫前期组超甲基化RASSF1A基因含量为正常同期妊娠组的3.52倍。结论:孕妇血浆中超甲基化的RASSF1A基因可以作为胎儿表观遗传学标志物,其水平的异常变化或能提示子痫前期-子痫的发生。

Objective： To investigate the feature of hypermethylated Ras association domain family 1A（RASSF1A） gene in maternal plasma and analyze the RASSF1A levels in pre-eclampsia.Methods： The methylation-sensitive restriction enzymes of HinP1I and HhaI were specifically cutting the maternally derived background hypomethylated sequences but leaving the intact hypermethylated sequences,a standard curve using serial dilutions of the standard preparation was employed,RASSF1A levels after the double enzymes digestion were determined using fluorescence quantitative polymerase chain reaction（FQ-PCR） to measure cell-free fetal DNA.At the same time,b-actin gene was detected as a control to confirm complete enzymes digestion.Results： 1） Fetal DNA can be detected in maternal plasma as early as at 7＋3th week of gestation;2） The concentrations of hypermethylated RASSF1A in maternal plasma increased as pregnancy progresses,and they were completely cleared at 24 h after delivery;3） Hypermethylated RASSF1A was not detected in the plasma of non-pregnant women;4）The mean concentrations of hypermethylated RASSF1A gene were 3.52-fold higher in maternal plasma of pre-eclampsia in 3rd trimester pregnancies than the controls.Conclusion： Hypermethylated RASSF1A gene may be considered as a epigenetic marker to detect the fetal DNA in maternal plasma,the elevated amounts of placenta-derived RASSF1A may forecast the process of pre-eclampsia and eclampsia.