用金纳米-分子信标的荧光定量PCR新技术研究6个miRNAs在心肌缺血患者中的表达

摘要

Objective To investigate the usage of a novel fluorescence real-time polymerase chain ration(RT-PCR) technology u-tilizing gold nanoparticle(AuNP) and molecular beacon(MB) probe for the quantitative detection of 6 microRNAs(miRNAs) in patients at different stage of ischemic cardiovascular disease.Methods The expression of the 6 miRNAs in 200 patients with ischemic cardiovascular disease were detected by using the sensitive detection method constructed in this study, and the results were validated by flexible multi-analyte profiling(xMAP).Results In patients group with acute myocardial ischemia,the serum levels of miRNA-21,miRNA-133,miRNA-199,miRNA-208 and miRNA-499 were significantly up-regulated in 1 - 3 hours after the onset of acute myocardial infarction.In patients group with chronic myocardial ischemia, the expression of miRNA-21, miRNA-133, miRNA-199 and miRNA-1 were significantly up-regulated in patients combined with primary hypertension and myocardial ischemia.And the expression levels of the miRNAs mentioned above were significantly higher in the two patients groups than those in control group(P <0.05,P<0.001).The specificity was 86.7% and the sensitivity was 90.2% of AuNP-MB assay,which were higher than 53.4% and 66.8% respectively,of MB assay.Conclusion Novel fluorescence RT-PCR assay using AuNP-MB technology, constructed in this study,could be used for the early detection of miRNAs and the early diagnosis of ischemic cardiovascular disease.%目的 探讨新建立金纳米-分子信标探针的实时荧光定量PCR技术及其分析不同程度心肌缺血患者的6个微小RNA(microRNA,miRNA):miRNA-1、miRNA-21、miRNA-133、miRNA-199、miRNA-208、miRNA-499的表达水平.方法 用新构建金纳米-分子信标探针引入实时荧光定量PCR技术分析200例不同程度心肌缺血患者血清中的以上6个miRNAs的表达水平,并用xMAP液态芯片技术验证以上miRNAs的表达.结果 急性心肌缺血组:在急性心肌梗死(AMI)发病后1～3 h检测到miRNA-21、miRNA-133、miRNA-199、miRNA-208、miRNA-499的表达上调;慢性心肌缺血组:通过对原发性高血压并发心肌缺血患者研究显示miRNA-21、miRNA-133、miRNA-199、miRNA-1的表达上调,以上两组分别与健康对照组比较差异有统计学意义(P<0.05,P<0.001).新构建的金纳米-分子信标探针检测的特异度为86.7%,灵敏度为90.2%,均高于常规分子信标(53.4%、66.8%),两种方法 比较差异有统计学意义(P<0.001),与xMAP液态芯片技术比较差异无统计学意义(P>0.05).结论 新建立的金纳米-分子信标探针的实时荧光定量PCR技术可作为早期检测miRNAs新的实验方法,为心肌梗死患者的早期诊断提供新的检测手段.