应用原位PCR检测母体循环中胎儿遗传信息HbF γmRNA的表达  

Detection of the expression of cellular fetal hemoglobin gamma chain messenger RNA(HbF-γmRNA) in maternal circulation for non-invasive prenatal diagnosis

作　　者：汤冬玲[1] 周新[2] 李艳[1] 郑芳[2] 李从荣[1] 

机构地区：[1]武汉大学人民医院检验科,430060 [2]武汉大学中南医院基因诊断中心,430071

出　　处：《中国优生与遗传杂志》2010年第1期16-18,56,共4页Chinese Journal of Birth Health & Heredity

基　　金：湖北省科技攻关计划项目(2003AA304B09)

摘　　要：目的原位PCR是一种直接在细胞或组织材料标本上原位扩增目的DNA或RNA片段,并在原位检测扩增产物,从而进行细胞内特定核酸序列检出及定位的分子技术。胎儿血红蛋白(fetal hemoglobin,HbF)(α2γ2)是胎儿红细胞中一种主要的胞浆蛋白,依据其特有的发育规律,拟从转录水平的角度,采用高灵敏度的反转录聚合酶链反应(RT-PCR)结合原位杂交技术,深入探讨胎儿血红蛋白γmRNA在孕妇外周血中的表达机理。方法取EDTA抗凝的孕妇静脉血5 ml,PBS稀释后,经淋巴细胞分离液分离获取单个核细胞层。PBS洗3次,离心后将沉淀涂片在经多聚赖氨酸处理的载玻片上,10%缓冲中性甲醛固定,PBS洗3次,蛋白酶K消化,PBS洗3次,气干。用地高辛标记的脱氧三磷酸尿苷掺入的原位RT-PCR的方法进行检测,经25轮PCR循环后,用碱性磷酸酶标记的抗地高辛抗体检测扩增产物,显色镜检,分析36例孕妇外周血中胎儿血红蛋白γ基因的表达情况。结果靶序列的原位RT-PCR结果显示,36例孕妇外周血中有30例标本中可定位检测到胞核、胞浆内的蓝紫色的阳性信号,符合率为83.33%,在孕妇外周血和初产妇的脐血的有核红细胞的胞浆内均可检测到DIG标记的HbF-γmRNA蓝紫色信号,而正常人外周血则无此信号。结论原位RT-PCR将PCR技术的高效扩增与细胞定位相结合,在细胞原位检测低拷贝mRNA,具有敏感性高、特异性强的特点,能从分子水平上定位定量检测HbF-γmRNA的表达,该基因仅在孕妇外周血中表达,未孕女性未见其表达,提示HbF-γmRNA可能为孕妇外周血中的一种新的靶标,为无创性产前诊断提供线索和思路。Objective： Fetal hemoglobin （HbF） is the predominant cytoplasmic protein found in fetal erythrocytes, thus this study aimed to explore the expression of fetal hemoglobin gamma chain messenger RNA （ HbF -γmRNA） as a convenient, sensitive and specific method for prenatal diseases. Methods： Maternal blood was obtained from 36 pregnant women at 13 to 38 weeks of gestation. NRBCs were separated with Pereoll using a discontinuous density gradient method, and the expression of fetal hemoglobin gamma chain messenger RNA （HbF -γ mRNA） was measured by in sitn RT-PCR in maternal peripheral blood. In situ PCR consisted of 15 cycles （ 1 rain at 94℃, 1 min at 62℃, 1 min at 72℃） followed by 7 rain at 72℃. After in situ PCR, the cells were washed twice with PBS, and DIG-labeled DNA was detected with a DIG Detection Kit （Roche Diagnostics）, according to the manufacturer＇s instructions. Cells were washed with PBS, mounted, and observed using a light microscope. The same procedures were done without including PCR primers for the negative control. Results ： DIG labeled HbF -γ, mRNA was detected in NRBCs from maternal peripheral and umbilical cord blood in situ RT -PCR method for 15 cycles. The signal was not, however, detected in leukocytes, red blood cells or reticulocyte. In non pregnant women, the signal was not detected in normal NRBCs. Conclusions ： It is indicated that HbF - γmRNA was detected in only fetal NRBCs. It was the first time we used non - invasive way to detect expression of fetal γ gene in maternal blood. Success of in situ RT - PCR to detect fetal specific mRNA gave the hint that the mRNA of the HbF -γ chain might be a sensitive fetal marker, the ways could be used in the field of prenatal diagnosis of hemoglobin disease, predicting fetal gender, and single gene disease and be used widespread in prenatal diagnosis.

关 键 词：原位PCR 胎儿血红蛋白 产前诊断 

分 类 号：R714.55[医药卫生—妇产科学]