实时荧光定量RT-PCR检测人外周血单个核细胞中FOXP3 mRNA的表达

Detection of FOXP3 mRNA expression in human peripheral blood mononuclear cells by real-time fluorescent quantitative RT-PCR

目的:建立实时荧光定量逆转录-多聚酶链反应(real-time fluorescence quantitative RT-PCR)检测人外周血单个核细胞中FOXP3 mRNA的方法,并研究其与CD4+CD25+Treg细胞活性相关性。方法:提取人外周血单个核细胞总RNA,将mRNA逆转录成cDNA,以β-actin为内参照,实时荧光定量RT-PCR检测29例哮喘患儿及24例同龄对照FOXP3 mRNA的相对表达量,采用融解曲线和琼脂糖电泳鉴定PCR产物特异性;同时采用流式细胞技术检测CD4+CD25+Treg细胞含量,分析两者相关性。结果:哮喘患儿组CD4+CD25+Treg细胞百分率明显低于同龄对照组,P<0.01;FOXP3 mRNA的表达也显著降低,P<0.05;FOXP3和β-actin的融解曲线分析表明均仅有单一峰,Tm值分别为82.4℃和87.8℃;琼脂糖电泳显示都仅有单一扩增产物。结论:应用SYBR GreenⅠ实时荧光定量RT-PCR技术检测FOXP3 mRNA表达水平简便易行、结果稳定可靠。初步结果证实,外周血CD4+CD25+Treg细胞数量和FOXP3 mRNA表达有相同的趋势,存在一定的相关性。

Objective：To establish a real-time fluorescent quantitative reverse transcription polymerase chain reaction（RT-PCR）to detect FOXP3 mRNA expression in human peripheral blood mononuclear cells（PBMCs）and to investigate the correlations between activity of CD4＋CD25＋ regulatory T cells and expression of FOXP3 mRNA.Methods：Total RNA was extracted with Trizol from human PBMCs and mRNA was transcribed reversely into cDNA.Real-time fluorescent quantitative RT-PCR with β-actin as the internal control gene was used to detect the expression levels of FOXP3 mRNA in 29 pediatric patients with asthma and 24 age-matched healthy children. The specificity of PCR productions was identified with the dissociation curves and agarose gel electrophoresis. Flow cytometry analysis was used to assess the percentage of CD4＋CD25＋regulatory T cells. The correlation between the expression and activity was analyzed. Results： The percentages of CD4＋CD25＋ regulatory T ceils in group of asthma were significantly lower than those in group of control （P〈0.01）, so did the expressions of FOXP8 mRNA （P〈O.O5）. The analysis of dissociation curves on FOXP3 and β-actin showed that both of them had only one simple spike and the Tmvalues were 82.4 ~C and 87.8 ~ ,respectively. The agarose gel electrophoresis of them showed only single PCR product each. Conclusion： It is an easy and reliable test to detect the expression level of FOXP8 mRNA by SYBR Green I real-time fluorescence quantitative RT-PCR and the preliminary experimental result shows that the percentages of CD4＋CD25＋ regulatory T ceils and the expressions of FOXP3 mRNA have the same trend and confirms the correlation between them.