【作者】 王健；

【导师】 赵富玺；

【作者基本信息】 山西医科大学 ， 免疫学， 2010， 硕士

【摘要】 目的：探讨应用TaqMan探针实时定量PCR技术检测孕妇血浆中微量胎源性Ras相关区域家族1A (ras association domain family 1A, RASSFIA)基因的可行性；对正常妊娠期间孕妇血浆中酶切前、后RASSFIA基因水平定量分析,且进一步证实酶切后RASSFIA基因具有胎儿DNA的性质,弥补遗传基因标志物的不足；探讨其含量变化在子痫前期中的应用价值。方法：选择妊娠7-41周孕妇80例,其中妊娠早、中、晚期及子痫前期孕妇各20例(轻、重度各10例),另取20例健康未妊娠女性为阴性对照。提取血浆游离DNA,经两种甲基化敏感的限制性内切酶HinP1I和Hhal消化处理,去除非甲基化的RASSFIA序列,保留超甲基化RASSFIA序列。应用基于TaqMan探针的荧光定量PCR (FQ-PCR)技术测定血浆中酶切前、后RASSFIA基因的含量,分别代表总游离DNA和游离胎儿DNA水平。同时检测β肌动蛋白(β-actin)基因,确保酶的完全消化。结果：(1)80例孕妇除3例早期孕妇外,其余均检出了酶切后RASSFIA基因,且最早可在妊娠7+3周检测出该基因；未妊娠对照组中酶切后均未检测到RASSFIA基因,检测孕妇血浆中超甲基化RASSFIA基因的灵敏度和特异度分别为96.25%和100%。(2)孕妇血浆中酶切后RASSFIA基因的含量随妊娠的进程逐渐增加,在妊娠期间,超甲基化RASSFIA基因的平均浓度在早期妊娠组为55.52拷贝／ml,中期妊娠组为112.46拷贝／ml,晚期妊娠组为557.50拷贝／ml,分别占酶切前总游离DNA的1.78%、2.91%、9.45%。(3)子痫前期组孕妇血浆中酶切后RASSFIA基因含量为正常同期妊娠组的3.72倍,且其水平变化与子痫前期病情的严重程度呈正相关。(4)10例正常晚期孕妇分娩后24小时均检测不到超甲基化RASSFIA基因,10例子痫前期孕妇分娩后24小时有8例仍能检测到该序列,在分娩后1周检测不到。结论：孕妇血浆中超甲基化RASSFIA基因具有游离胎儿DNA的性质,是一种胎儿特异性表观遗传学标记物；应用Taqman探针荧光定量PCR技术可以更简便、准确地检测孕妇血浆中微量胎源性RASSFIA序列的水平；孕妇血浆中超甲基化RASSFIA基因浓度变化与子痫前期的病情具有相关性,其含量的异常变化可以提示子痫前期-子痫的发生及预后,扩大了游离胎儿DNA在无创产前诊断中的应用范围。更多还原

【Abstract】 Objective:To investigate the feasibility of quantitative detection of hypermethylated Ras association domain family 1A (RASSFIA) gene in maternal plasma by TaqMan probe real-time PCR and analysis the RASSFIA levels before and after methylation-sensitive restriction enzymes digestion. The aims of the detection of hypermethylated RASSFIA gene in maternal plasma from all three trimesters of pregnancy were to show its feature of cell free fetal DNA and to make up defieients of genetie markers. This study also aimed at investigating of its application value in pre-eclampsia.Methods:80 pregnant women(7-41 gestational weeks) including pregnancy of first trimester (20 cases), second trimester (20 cases), third trimester (20 cases), mild pre-eclampsia (10 cases) and severe pre-eclampsia (10 cases) were selected as study groups,20 normal non-pregnant women were selected as control group. Free DNA of plasma samples was extracted, The methylation-sensitive restriction enzymes were specifically cutting the maternally derived background hypomethylated sequences but leaving the intact hypermethylated sequences, RASSFIA levels before and after double methylation-sensitive restriction enzymes digestion of HinP1I and Hhal were determined using fluorescence quantitative polymerase chain reaction (FQ-PCR) to measure total and fetal cell-free DNA, respectively. At the same time,β-actin gene was detected as a control to confirm complete enzymes digestion.Results:(1) Hypermethylated RASSFIA sequences in maternal plasma were detected in 77 of 80 pregnant women, and were not detected in the plasma of non-pregnant women. They can be detected in maternal plasma as early as at 7+3 th week of gestation. The sensitivity and specificity of fetal gene detection by post-digestion RASSFIA measurement were 96.25% and 100%. (2) The concentrations of hypermethylated RASSFIA in maternal plasma increased as pregnancy progresses, the mean concentrations of hypermethylated RASSFIA gene were 55.52copies/ml in first trimester,112.46copies/ml in second trimester,557.50copies/ml in third trimester, respectively. We futher observed the post-digestion RASSFIA concentrations as a fraction of the total RASSFIA concentrations obtained without enzymes digestion RASSFIA which corresponded to1.78%,2.91%, and 9.45% in the three trimesters. (3) The mean concentrations of hypermethylated RASSFIA gene were 3.72-fold higher in maternal plasma of pre-eclamptic pregnancies than in controls. There was positive correlation between fetal-derived hypermethylated RASSFIA levels and the severity of pre-eclampsia. (4) The post-digestion RASSFIA was completely cleared at 24h after delivery in 10 pregnant women of the third trimester, it still can be detectable in 8 of 10 pre-eclamptic pregnant women, which was cleared after a week.Conclusion:Hypermethylated RASSFIA gene may be considered as a epigenetic marker to detect the fetal DNA in maternal plasma. It is easy and precise to detect the hypermethylated RASSFIA gene in maternal plasma by TaqMan probe real-time PCR. Our study shows the correlation of maternal plasma hypermethylaed RASSFIA concentrations with pre-eclampsia. The elevated amounts of maternal plasma hypermethylaed RASSFIA can forecast the process and prognosis of pre-eclampsia and eclampsia. The quantitative detection of the placental epigenetic signature of the RASSFIA gene in maternal plasma expand the clinic application of cell free fetal DNA in noninvasive prenatal diagnosis.更多还原