Establishment of A Non-invasive Prenatal Diagnosis Method for Chromosomal Abnormalities

本研究证实，胎儿造血祖细胞在妊娠期和产褥期妇女外周血中的循环频率较高。1）研究对象为15例足月产男婴的妇女。在分娩后1小时内、4天和1个月时抽取3 - 9 ml外周血。通过Ficoll-hypaque离心从这些血液样品中分离的单核细胞在含有PHA-LCM和促红细胞生成素的甲基纤维素培养基中培养。培养2至3周后，计数培养基中形成的菌落，并将其单独收集到PCR管中的低渗溶液中。煮沸法提取DNA，巢式PCR法同时扩增Y染色体SRY基因和7号染色体ZP 3基因的DNA片段。扩增产物在琼脂糖凝胶上电泳并通过溴化乙锭染色可视化。我们可以区分来自胎儿造血祖细胞的集落 ...更多信息 从分娩当天采集的1 ml外周血中分离的单核细胞（n=13）总共形成42.7 × 19.6（平均值× 1 SD）集落。进一步的PCR扩增显示，每1 ml母体血液中显示雄性带型的胎儿衍生集落的数量为4.8 × 2.4。2）对17例产男婴的孕妇进行了同样的菌落分析，其中2例在产后4天（n=2）和5例在产后1个月（n=5）分别检测到7.5 × 2.9和1.1 × 1.1个菌落。采集孕14 ~ 35周外周血。胎儿驱动集落的频率为3.7* 2.3%，转换为4.1*3.1（范围0-10.0）/1 ml母体血液。每1 ml母体血液中的胎儿菌落数与胎龄之间存在显著相关性（R=0.58，p=0.015）。目前正在对妊娠早期的病例进行分析。上述结果表明，我们可以从母体血液中收集显示100%纯度和增殖能力的胎儿细胞，使用这些胎儿细胞进行随后的细胞遗传学分析是毫不费力的。因此，我们建议，我们可以建立非侵入性的大规模筛选方法，染色体异常的推广和简化我们的分析系统。少

We verified that fetal hematopoietic progenitor cells circulate with a significant frequency in peripheral blood of women in pregnancy as well as puerperium period, as follows.1) The objects were 15 women who were delivered of male infants at term. Three to 9 ml of their peripheral blood was drawn within 1 hour, at 4 days, and at 1 month post-delivery. Mononuclear cells separated from these blood samples by Ficoll-hypaque centrifugation were cultured in methylcellulose medium containing PHA-LCM and erythropoietin. After 2 to 3 weeks of culture, colonies formed in medium were counted and individually collected into hypo-osmotic solution in PCR tubes. DNAs were extracted by boiling, and DNA fragments in the SRY gene on Y chromosome and the ZP3 gene on chromosome 7 were simultaneously amplified by nested PCR method. Amplified products were electrophoresed on agarose gels and visualized by ethydiumbromide staining. We could distinguish the colonies derived from fetal hematopoietic progenit … More ors by the presence of both the SRY and ZP3 bands.Mononucleated cells separated from 1 ml of peripheral blood taken on delivery day (n=13) formed 42.7*19.6 (mean *1 SD) colonies in all. Further PCR amplification revealed that the number of fetus-derived colonies exhibiting the male band pattern was 4.8*2.4 per 1 ml of maternal blood. 7.5*2.9 and 1.1*1.1 of fetal colonies were also detected in the women at 4 days (n=2) and I month (n=5) after their deliveries, respectively.2) The same colony analysis was applied to 17 pregnant women having male infants. Their peripheral blood was drawn from 14 to 35 weeks of gestation. The frequency of fetus-drived colonies was 3.7*2.3%, which was converted to be 4.1*3.1 (range 0-10.0) per 1 ml of maternal blood. There was a significant correlation between fetal colony numbers per 1 ml of maternal blood and gestational age (R=0.58, p=0.015). The analysis of cases in the first trimester is under way. The above results indicated the possibility that we can collect fetal cells exhibiting 100 % of purity and ability of prolifiration from maternal blood, It is effortless to perform subsequent cytogenetic analysis using these fetal cells. Therefore, it was suggested that we could establish noninvasive mass-screening method for chromosome abnormalities by generalizing and facilitating our analysis system. Less