孕妇外周血中胎盘特异性外泌体DNA作为胎儿染色体异常的分子标记物的研究

During pregnancy, cell-free DNA and placenta-derived exosomes are released into maternal circulation from human placental trophoblast cells. The distribution of cell-free DNA and placenta-derived exosomes in maternal circulation shares similar association with gestational age, maternal condition, and placental pathology. Therefore we speculate that some cell-free DNA is packaged in the placental exosomes and enter maternal circulation. The aim of this project is to prove the existence of cell-free DNA in placental exosomes obtained from maternal blood, and to evaluate the placental exosomes DNA as the biomarker of fetal chromosome abnormality. We aim to develop in vivo experiments to extract placental exosomes from maternal plasma using magnetic beads conjugated with specific antibodies. Next generation sequencing is then used to sequence the placental exosome DNA to analyze the molecular characteristics such as chromosomal coverage, fetal fraction, and GC bias. Placental exosome DNA from pregnant women carrying fetus with trisomy or thalassemia will be sequenced, and bioinformatics pipelines will be developed, in order to identify chromosomal abnormality or disease mutation. The knowledge gained from this project could be used to develop novel prenatal testing methods on the basis of placental exosome DNA sequencing. It may also improve our understanding of the biological functions of placental exosomes in normal and pathogenic condition during pregnancy, and the mechanism of the generation and turnover of cell-free DNA.

妊娠期间，人类胎盘滋养层细胞存在向母体循环释放游离DNA与释放胎盘特异性外泌体两种现象，并且它们都与孕周、母体生理条件、胎盘功能有紧密联系，存在一定的含量变化相似性。据此，我们推测部分游离DNA通过外泌体转运途径从胎盘滋养层细胞进入母体循环。本项目拟从孕妇外周血中提取胎盘特异性外泌体，验证其含有胎源游离DNA的假说，并评估胎盘特异性外泌体DNA作为胎儿染色体异常的分子标记物的可能性。通过建立免疫磁珠提取方法，可以获得真实反映体内妊娠条件的胎盘特异性外泌体，进而使用高通量测序技术研究胎盘特异性外泌体DNA的基因组分布、胎儿浓度、GC偏好性等分子特征。通过使用胎盘特异性外泌体DNA检测染色体非整倍体和地中海贫血致病基因，开发相应的信息分析方法，本项目可以为发展基于外泌体DNA的检测新技术奠定基础，为了解胎盘特异性外泌体在正常和异常妊娠生理条件下的功能、游离DNA的产生与体内更迭机制提供参考依据

妊娠期间孕妇外周血中存在来自胎盘滋养层细胞释放的游离DNA和外泌体，二者存在一定的含量变化相似性，并且都与孕周、母体生理条件、胎盘功能有紧密联系。既往研究显示肿瘤细胞外泌体中存在包括DNA在内的多种信号分子。因此，本研究拟从孕妇外周血中提取胎盘特异性外泌体，验证其含有胎源游离DNA的假说，并评估外泌体DNA作为胎儿染色体异常和单基因病的分子标记物的可能性。.本项目研究内容包括建立从孕妇血浆中提取胎源外泌体的方法，利用qPCR和高通量测序等方法鉴定外泌体中含有的DNA片段的分子特征，开发生物分析流程对外泌体DNA高通量测序数据进行分析，验证其用于检测胎儿染色体三体和单基因病的可行性。.本项目采用了多种不同方法对20例正常孕妇血浆分别提取外泌体，经过透射电镜和western blotting检测确认可从孕妇血浆中提取到具有典型形态和表面蛋白标记物的外泌体。项目组利用DNA提取试剂盒提取了外泌体DNA，进而采用qPCR方法准确检测到男胎的Y染色体信号，并进行男女胎区分，证明了孕妇血浆中的外泌体含有胎源DNA。为了鉴定外泌体DNA的分子特征，我们对外泌体DNA进行了高通量测序和生物信息分析，结果显示孕妇血浆外泌体DNA和血浆游离DNA类似，呈全基因组分布，但外泌体DNA的GC含量和线粒体DNA比例较游离DNA分别高出1.16倍和2.18倍。外泌体DNA的片段为152.4±10.51bp，小于游离DNA的 168.5±1.25bp。此外，外泌体DNA并未显示出胎儿浓度富集的现象。因此外泌体DNA与血浆游离DNA相比，既有相似性也有差别。利用高通量测序对13例三体胎儿孕妇和2例软骨发育不全胎儿孕妇的血浆外泌体DNA进行检测，可以准确检测到胎儿的21、18、13-三体，以及造成软骨发育不全的FGFR3基因点突变，而在20例正常孕妇血浆外泌体DNA中不会出现假阳性结果，证明可以通过外泌体DNA检测胎儿染色体异常和单基因病。综上，证明了孕妇血浆中存在包含胎源DNA片段的外泌体，并首次利用高通量测序的方法鉴定了外泌体DNA的分子特征，验证了其用于胎儿遗传疾病分子标记物的可行性。