Yagel S, Shpan P, Dushnik M, Livni N, Shimonovitz S
Department of Obstetrics and Gynaecology, Hadassah Mount Scopus, Jerusalem, Israel.
Hum Reprod. 1994 Jun;9(6):1184-9. doi: 10.1093/oxfordjournals.humrep.a138656.
Trophoblasts have been detected in uterine venous blood, lung parenchyma and maternal blood in the first trimester. Their dilution within maternal leukocytes has been recently estimated to be 1:10(-6). The objectives of this study were to enrich peripheral maternal blood preparations for trophoblast cells, to isolate trophoblasts from the enriched preparation by highly specific markers and to assess fetal cell total number of chromosomal copies by fluorescence in-situ hybridization (FISH). Negative and positive selections for trophoblasts were performed. To assess the efficacy of the enrichment methods, a model mimicking the in-vivo conditions was established. Purified first trimester trophoblasts were prepared from first trimester placentas and were mixed with leukocytes from non-pregnant women in various concentrations. Magnetic beads coupled with antibodies to the common leukocyte antigen (CD45) or to antitrophoblast specific antigens (Trop1, Trop2 and GB25), were attached to peripheral maternal blood cells or to the prepared mixed cell populations. The expression of alpha human chorionic gonadotrophin (alpha HCG) or of human placental lactogen (HPL) by the remaining cells was examined by two means: (i) immunocytochemistry, using monoclonal antibodies against HPL and alpha HCG to stain fetal cells; (ii) reverse transcriptase polymerase chain reaction (RT-PCR), using specific primers for exons of the HPL or alpha HCG mRNAs. Results revealed that HPL- and alpha HCG-expressing cells could be identified in maternal blood only in very rare instances. On the other hand, expression of alpha HCG and HPL by only 100 purified first trimester trophoblasts artificially mixed with peripheral leukocytes at a ratio of 1:10(-5) could be identified by both immunocytochemistry and RT-PCR.(ABSTRACT TRUNCATED AT 250 WORDS)
在孕早期，已在子宫静脉血、肺实质和母体血液中检测到滋养层细胞。最近估计它们在母体白细胞中的稀释度为1:10⁻⁶。本研究的目的是富集母体外周血中的滋养层细胞，通过高特异性标志物从富集的样本中分离滋养层细胞，并通过荧光原位杂交（FISH）评估胎儿细胞染色体拷贝的总数。对滋养层细胞进行了阴性和阳性选择。为评估富集方法的有效性，建立了一个模拟体内条件的模型。从孕早期胎盘制备纯化的孕早期滋养层细胞，并将其与不同浓度的未孕女性白细胞混合。将与常见白细胞抗原（CD45）抗体或抗滋养层特异性抗原（Trop1、Trop2和GB25）偶联的磁珠附着于母体外周血细胞或制备的混合细胞群体上。通过两种方法检测剩余细胞中α人绒毛膜促性腺激素（α HCG）或人胎盘催乳素（HPL）的表达：（i）免疫细胞化学，使用抗HPL和α HCG的单克隆抗体对胎儿细胞进行染色；（ii）逆转录聚合酶链反应（RT-PCR），使用HPL或α HCG mRNA外显子的特异性引物。结果显示，仅在极少数情况下才能在母体血液中鉴定出表达HPL和α HCG的细胞。另一方面，通过免疫细胞化学和RT-PCR均可鉴定出仅100个纯化的孕早期滋养层细胞与外周白细胞以1:10⁻⁵的比例人工混合时α HCG和HPL的表达。（摘要截短至250字）
