应用密度梯度离心结合免疫磁珠法富集孕妇外周血中胎儿NRBC的实验研究

Enrichment of fetal nucleated red blood cells from maternal peripheral blood by using density gradient centrifugation and magnetically activated cell sorting

目的研究利用不连续密度梯度离心结合免疫磁珠方法,分选孕妇外周血中的胎儿有核红细胞(NRBC)。方法采集21名孕周在12-28周的孕妇外周血10ml,经不连续密度梯度离心后,再用免疫磁珠法分选和富集胎儿NRBC,同时对富集前后的孕妇外周血进行瑞氏染色和胎儿血红蛋白(fetal hemoglobin,HbF)特异性抗体标记、识别。结果未经分选、富集,21名孕妇外周血有核细胞层涂片经瑞氏染色检查均未发现NRBC;梯度离心富集后21名孕妇中有11例外周血有核细胞涂片找到1～8个NRBC;再经免疫磁珠分离后有12例涂片发现1～7个NRBC,同时进行HbF特异性抗体标记,证实其NRBC阳性涂片中均存在胎源性NRBC。结论本研究初步建立了分选、富集胎儿NRBC的实验方法,通过进一步的方法学改进,有望用于产前基因筛查的研究。

Objective To sort and enrich fetal nucleated red blood cells （NRBCs）from maternal peripheral blood by using discontinuous density gradient centrifugation （DGC） and magnetically activated cell sorting （MACS）. Methods A total of 21 pregnant women with a gestation of 12 to 28 weeks were studied in this paper, 10 ml peripheral blood was obtained froln each pregnant woman, NRBCs were sorted by methods of DGC and MACS. Meanwhile, smear slides of maternal peripheral blood before and after enrichment step were subjected to Wright＇s staining and the staiming of antibody against the fetal henloglobin （ltbF）. Results No NRBCs was found in the Wright＇s staining slides of maternal blood without enriching NRBCs, Enrichment of NRBCs by DGC method, there were 1-8 NRBCs which were fouild in 11 of 21 maternal samples; Combined DGC method with MACS, there were 1-7 NRBCs in 12 of 21 maternal samples respectively. And the staining results using antibody, against HbF confirmed that fetal NRBCs exist in the slides. Conclusion Our study had primarily established the efficient method of sorting and enrichment of fetal NRBCs from maternal blood, it can bc applied in the further prenatal genetic diagnosis and other research work.