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

我们证实胎儿造血祖细胞在妊娠期和产褥期妇女外周血中循环频率显著，如下所示。研究对象是15名足月生下男婴的妇女。在分娩后1小时、4天和1个月抽取3 - 9ml外周血。用Ficoll-hypaque离心分离法分离的单个核细胞在含有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 ~ 3周后，计数培养基中形成的菌落，并将其单独收集到PCR管的低渗透溶液中。煮沸提取DNA，用巢式PCR法同时扩增Y染色体上SRY基因和7号染色体上ZP3基因的DNA片段。扩增产物在琼脂糖凝胶上电泳，并通过二溴化乙酯染色显示。我们可以通过SRY和ZP3条带的存在来区分来自胎儿造血后代的菌落。分娩当天取外周血1 ml分离的单核细胞（n=13），共形成42.7*19.6个（平均*1 SD）菌落。进一步的PCR扩增显示，胎儿来源的菌落显示雄性带型，每1 ml母体血液中有4.8*2.4个。分娩后第4天（n=2）和第1个月（n=5）分别检测到7.5*2.9和1.1*1.1个菌落。2)同样的菌落分析应用于17名有男婴的孕妇。在妊娠14 ~ 35周抽取外周血。胎儿驱动菌落的频率为3.7*2.3%，换算为每1 ml母体血液4.1*3.1个（范围0-10.0）。每ml母体血液中胎儿菌落数与胎龄有显著相关性（R=0.58, p=0.015）。目前正在对妊娠头三个月的病例进行分析。以上结果表明，我们有可能从母体血液中收集到纯度和增殖能力达到100%的胎儿细胞，使用这些胎儿细胞进行后续的细胞遗传学分析也很容易。因此，我们建议通过推广和完善我们的分析系统，建立无创的染色体异常大规模筛查方法。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