微小核糖核酸荧光定量聚合酶链反应检测方法的建立及其在急性肺损伤中的表达分析  

Stem-loop qRT-PCR to detect microRNAs expression in lipopolysaccharides-induced acute lung injury

作　　者：才志刚[1] 张绍明[1] 张珩[1] 周宜勇[1] 吴海波[1] 徐小平[1] 

机构地区：[1]中国人民解放军第四五五医院心胸外科,上海200052

出　　处：《上海医学》2011年第9期682-685,F0003,共5页Shanghai Medical Journal

基　　金：2009年南京军区医学科研立项课题基金资助(09MA028)

摘　　要：目的探讨微小核糖核酸(miRNA)荧光定量聚合酶链反应(PCR)检测方法的建立条件,并运用这一方法对急性肺损伤中miRNAs的表达进行分析。方法设计miRNA特异的颈环结构反转录引物,对miRNA进行反转录,得到miRNA的环状脱氧核糖核酸(cDNA)。分析miRNA cDNA定量PCR过程中的标准曲线和熔解曲线,确定该检测方法的检测灵敏性和特异性。应用miRNA的荧光定量PCR检测方法分析脂多糖(LPS)诱发的急性肺损伤小鼠中miRNA的表达。结果 miRNA特异的颈环结构反转录引物实现了miRNA长度的延长。通过对心肌特异表达的miRNA-1进行实时荧光定量PCR检测,表明miRNA-1是心肌组织特异表达的miRNA。通过该方法对LPS诱发的急性肺损伤小鼠(急性肺损伤组)进行分析,发现与对照组比较,急性肺损伤组的miRNA-23a的相对表达量显著降低(P<0.05),miRNA-155、miRNA-451的相对表达量显著升高(P值均<0.01),miRNA-21的相对表达量未发生改变(P>0.05)。结论本实验建立的miRNA实时荧光定量PCR具有较高的精确性和特异性,通过该方法发现多种miRNA在LPS诱发的急性肺损伤中异常表达。Objective To develop a novel microRNA quantification method to detect the expression level of microRNAs in lipopolysaccharides （LPS）-induced acute lung injury. Methods Stem-loop primers for specific miRNAs were designed and synthesized for reverse transcription reaction. After the amplification, the obtained miRNA was transcribed to cDNA. SYBR was used for the real-time polymerase chain reaction （PCR） analysis for the miRNAs expression level. The dynamic range and melt curve of qRT-PCR were verified to evaluate the sensitivity and specificity of this method. After mouse model of lung injury was established by LPS intratracheal administration, qRT-PCR was used to measure the expression profiles of miRNAs in the heart, liver, lungs and kidneys of the mice. Results The designed stem-loop primers successfully amplified miRNA. Among the four tissues examined, microRNA-1 （miRNA-1）, a muscle-specific microRNA, was only detected in the heart. Realtime PCR analysis showed that miRNA-23a was down-regulated （ P〈0.05）, miRNA-155 and miRNA-451 were upregulated （both P〈0.01 ）, and miRNA-21 was not altered （P〈0.05） in LPS-induced lung injury when compared with the negative model. Conclusion The method we established has high accuracy and specificity analyzing of microRNAs expression. With this method, many microRNAs show abnormal expression profiles in mice with LPS- nduced acute lung injury. （Shanghai Med J, 2011, 34： 682-685）

关 键 词：微小核糖核酸 A549肺腺癌细胞株 脂多糖 急性肺损伤 颈环结构 

分 类 号：R563.8[医药卫生—呼吸系统]