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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。
Purpose In situ PCR is a molecular technique that directly amplifies a DNA or RNA fragment of interest in situ on a cell or tissue specimen and detects the amplification product in situ to detect and locate specific nucleic acid sequences within the cell. Fetal hemoglobin (α2γ2) is a major cytoplasmic protein in fetal erythrocytes. Based on its specific developmental rules, it is proposed that high sensitivity RT-PCR (RT-PCR) -PCR) combined with in situ hybridization to investigate the expression of fetal hemoglobin γmRNA in peripheral blood of pregnant women. Methods EDTA anticoagulant pregnant women venous blood 5 ml, diluted with PBS, mononuclear cell layer obtained by lymphocyte separation liquid separation. PBS washed three times, the sediment after centrifugation the smear on polylysine treated glass slides, 10% buffered neutral formaldehyde, washed with PBS 3 times, digested with Proteinase K, PBS washed 3 times, air-dried. Digoxigenin-labeled deoxynucleoside triphosphate incorporation in situ RT-PCR method for detection, after 25 cycles of PCR, with alkaline phosphatase labeled anti-digoxin antibody detection of amplification products, color microscope Detection, analysis of 36 cases of pregnant women with fetal hemoglobin γ gene expression. Results The RT-PCR results of target sequence showed that the positive signals of blue-purple in nucleus and cytoplasm were detected in 30 specimens of peripheral blood of 36 pregnant women with the coincidence rate of 83.33%. In the peripheral blood of pregnant women DIG-labeled HbF-γmRNA blue-violet signal was detected in cytoplasm of nucleated red blood cells of umbilical cord blood of primipara, while normal human peripheral blood did not. Conclusion In situ RT-PCR combines the efficient amplification of PCR with cell localization to detect low-copy mRNA in situ in cells. It has the characteristics of high sensitivity and specificity, and can quantitatively detect HbF-γ mRNA at the molecular level The gene was expressed only in the peripheral blood of pregnant women, but not in the non-pregnant women, suggesting that HbF-γmRNA may be a new target in the peripheral blood of pregnant women, providing clues and ideas for noninvasive prenatal diagnosis.