摘要
目的探讨Keihuaer抗酸染色法标记孕妇外周血中胎儿有核红细胞（fetal nucleated red blood cell，FNRBC），并结合单细胞聚合酶链式反应（polymerase chain reaction，PCR）技术行产前性别相关基因诊断的可行性。方法采集12例孕周为10周-16周的孕妇外周血10ml，经Percoll不连续密度梯度离心初步分离后，运用Keihuaer抗酸染色法对FNRBC进行特殊染色标记，显微操作法获取阳性单个靶细胞，经过多重置换扩增，全基因组扩增产物作为PCR模板，行STS（steroid sufatase）基因同源序列PCR性别诊断，以验证靶细胞的胎源性。结果12例孕妇外周血中FNRBC阳性标记11例，1例未获取FNR-BC；对获取的4个FNRBC单细胞进行性别初步诊断，其中2例证实为男胎，2例为女胎，经证实均与临床诊断相符合，准确率为100％。结论用Keihuaer抗酸染色标记及显微操作法获取的FNRBC，经多重置换扩增后可用于无创性产前基因诊断，实验操作可行、诊断准确，具有重要临床应用价值。
Objective To investigate the possibilities of using the Keihuaer acid - fast staining method to mark fetal nucleated red blood cell（FNRBC） in peripheral blood of pregnant woman, as well as utilizing the single -cell polymerase chain reaction（PCR） technique to make prenatal diagnosis related to sex gene. Methods About ten milliliter of peripheral blood sample from every twelve pregnant women with gestational weeks arranged from ten to sixteen was colleted, and then subjected to the discontinuous density gradi- ent centrifugation by using percoll reagent. After the primary cell isolation, Keihuaer acid - fast staining method was used to mark the isolated special cells previously spread on the slides. After gaining the single positive cell by the aid of microscopic manipulation tech- nique, the whole genome of single cell was amplified by using multiple displacement amplification（MDA） . The products were used as the template to further amplify the homologous steroid sulfatase gene sequences on X and Y chromosome respectively, so as to make a sex diagnosis and validate the fetus - originated characteristics of the target cell. Results The positive FNRBC were detected in eleven of twelve healthy pregnant women, no positive FNRBC was found in one of samples. Four positive FNRBC were picked out and subjected to primary sex diagnosis, two single cells were validated to be male origin, and the other two to be female origin. The diagno- sis results were entirely coincident with the clinical results. The diagnostic accuracy was 100%. Conclusion Using the Keihuaer acid-fast staining and microscopic manipulation technique to gain the single positive FNRBC was realistic, the latter was further subjec- ted to the MDA and polymerase chain reaction to make a non -invasive prenatal gene diagnosis. The whole experiment operation was feasible and the diagnosis results was accurate. It would hopefully prove its important worth in the clinical application.
出处
《中国计划生育和妇产科》
2009年第3期27-31,共5页
Chinese Journal of Family Planning & Gynecotokology
基金
安徽省科技攻关资助项目（项目编号：06013125B）
关键词
Keihuaer抗酸染色
胎儿有核红细胞
多重置换扩增
无创性产前基因诊断
Keihuaer acid - fast staining method
fetal nucleated red blood cell
multiple displacement amplification
non -invasive prenatal gene diagnosis
