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_临床医学论文.docVIP

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_临床医学论文 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_临床医学论文 作者：蒋卫平，丁茂文，朱亚非，李冰，朱晚林，李欣华，林菲菲 【摘要】 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。 【关键词】 实时荧光定量RT-PCR；哮喘；FOXP3；CD4+CD25+调节性T细胞 　　Abstract: 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 cells in group of asthma were significantly lower than those in group of control (P lt;0.01)， so did the expressions of FOXP3 mRNA (P lt;0.05). The analysis of dissociation curves on FOXP3 and β-actin showed that both of them had only one simple spike and the Tm values were 82.4 ℃ and 87.8 ℃，respectively. The agarose gel electrophoresis of