应用逆转录-聚合酶链反应方法检测孕妇外周血中胎儿ε/γ血红蛋白基因

Expression of fetalεandγglobin gene in maternal peripheral blood

目的 探索检测孕妇外周血中胎儿特异血红蛋白基因表达的方法。方法 提取孕妇全血mRNA ,经体外逆转录后 ,用逆转录 聚合酶链反应 (RT PCR)方法 ,应用ε/γ特异引物引导扩增胎儿特异血红蛋白基因片段 ,经琼脂糖凝胶电泳后 ,在凝胶成像系统下观察。结果 在 7名妊娠妇女中 ,6名扩增出ε/γ基因片段 ,1名为阴性。结论 应用RT PCR方法可直接从孕妇全血中扩增出胎儿特异血红蛋白基因片段 ,孕妇全血中微量的胎儿mRNA已基本满足体外逆转录及扩增的要求。

Objective Traditional prenatal diagnosis for congenital diseases were villus sampling and amniocentesis. These invasive diagnosis methods are not only technical complicated, but also harmful to mother or fetus. Fetus in its different gestational age has its different type of hemoglobin or different amount of hemoglobin, especially ε hemoglobin exiting in the body of 10 weeks gestation fetal, however γ hemoglobin has its high amount before baby to be born. But ε and γ hemoglobin did not exist in the bodies of adults bodies. It is possible to use advanced molecular biological technique to extract the fetal hemoglobin gene from maternal peripheral blood. In articles from domestic and abroad, no report related to fetal hemoglobin extraction from maternal peripheral blood was found. We tried to use non invasive method to detect fetal hemoglobin ε/γ gene from maternal peripheral blood by molecular biological technique. The purpose was to establish a convenient, sensitive and special method to be a basis of screening prenatal diseases in the population and lay a basis for family planning and clinical application.Methods Blood samples were collected and the fetal mRNA extracted from the pregnant women with the use of random primer. We used ultraviolet spectrophotometer to test the concentration and purity of extracted mRNA are suitable for reverse transcription. Reverse transcription of mRNA into cDNA was carried out and cDNA by PCR with the special ε/γ primer being used. Via 1.2 % EB in agarose gel electrophoresis, we used 'Gel Works System' to scan the electrophoresis image to detect ε/γ gene band.Results The peripheral blood of pregnant women was collected. With RT PCR and agarose gel electrophoresis method, we detected ε/γ gene successfully in 7 samples with 6 positive and 1 negative. Conclusion This was the first time that we used non invasive way to detect expression of fetal ε/γ gene in maternal blood to have found that this was a simple method to separate fetal cells from maternal blood, and could easily be accepted by pregnant women. Success of RT PCR to detect fetal specific mRNA gave the hint that this method could be used in the field of prenatal diagnosis of hemoglobin disease, predicting fetal gender, predicting Rh blood type and single gene disease and be used widespread in prenatal diagnosis.