密度梯度离心法富集孕妇外周血中胎儿有核红细胞的方法学

Enriching feta nucleated erythrocytes from maternal blood by density gradient centrifugation

目的 探讨密度梯度离心法 (density gradient centrifugation,DGC)富集孕妇外周血中胎儿有核红细胞的可行性及其在产前基因诊断中的价值。 方法 采用密度梯度离心法富集孕妇外周血中胎儿有核红细胞 ,以流式细胞分析鉴定富集细胞 ,同时对富集前后孕妇外周血 DNA进行男性性别决定基因 (sex-determining region Y,SRY)检测。 结果 5例孕妇外周血单个核细胞靶细胞群糖蛋白 A (glycophorin A,GPA)阳性细胞富集前平均含量为 0 .0 2‰～ 0 .1‰ ,而富集后平均含量高达 0 .5%～ 2 .8% ;2 4例孕 10～ 40周孕妇 (包括以上 5例 )中 ,孕妇外周血 DNA富集前 SRY基因男性诊断符合率 69.2 % (9/ 13 ) ,诊断总符合率 83 .3 % (2 0 / 2 4) ,而富集后分别为 84.6%(11/ 13 ) ,91.6% (2 2 / 2 4) ,11例怀女胎的孕妇富集前、后均未检出 SRY基因。 结论 密度梯度离心法可从孕妇外周血中富集胎儿细胞 ,且所得细胞已基本满足体外扩增单拷贝基因所需的模板量 。

Objective To investigate the efficacy of Density Gradient Centrifugation(DGC) in enriching fetal nucleared erythrocytes from maternal blood and its role in Non invase prenatal gene diagnosis. Methods Fetal nucleated erythrocytes from maternal blood were enriched by DGC and examined by flow cytometry (FCM).Meanwhile,sex determining region Y(SRY) of maternal peripheral blood DNA before and after enrichment was amplified. Results The average amount of glycophorin A(GPA) positive cells of single nucleus target cells in 5 pregnant women was 0 02 %～0 1 ‰,and 0 5 %～2 8 % after enrichment;24 DNA samples extracted from maternal peripheral blood,including 13 samples from male bearing and 11 samples from female bearing pregnancies ranging from the 10 th to the 40 th gestational week,were amplified by nested PCR of the Y specific sequence SRY.In result,SRY gene was detected in 9 of 13 samples from male bearing before enrichment,whereas in 11 of 13 samples after enrichment.SRY gene was not detected in 11 samples from male bearing before and after enrichment. Conclusion DGC can enrich fetal uncleated erythrocytes from maternal blood.Fetal single copy gene can be determined by nested PCR amplification of Y chromosome specific DNA with these fetal cells.