实时荧光定量PCR检测人IL9 mRNA方法的建立及应用_临床医学论文.docVIP

实时荧光定量PCR检测人IL方法的建立及应用_临床医学论文 实时荧光定量PCR检测人IL方法的建立及应用_临床医学论文 作者：杨先知 史烨 柳迎昭 陈建国 田洁 　 刘艳 刘青玲苏兆亮 仝佳 马斌 马洁 邵启祥 许化溪 王胜军 【摘要】 目的： 建立检测人白介素，IL的SYBR GreenⅠ实时荧光定量PCR( real方法。方法： 分离人外周血单个核细胞(peripheral blood mononuclear cells, PBMC)，经PMA和离子霉素刺激活化后提取总RNA并逆转录成cDNA。扩增产物与pMD载体连接构建质粒标准品。采用qRT方法检测IL的表达水平。评价该方法的线性及特异性，应用该方法检测Graves病患者PBMC中IL表达水平。结果： 建立了人IL的SYBR GreenⅠ实时荧光定量PCR检测方法。该法的标准曲线相关系数r2为0.995～0.998，熔解曲线分析产物为单峰。Graves病患者PBMC中IL的相对表达水平明显高于健康对照组(0.01)。结论： 建立的检测人IL的SYBR GreenⅠ实时荧光定量PCR方法灵敏、特异性好，为进一步临床应用提供了实验基础。 【关键词】 白细胞介素实时荧光定量PCR; SYBR GreenⅠ 　　[Abstract] Objective: To establish a SYBR GreenⅠ based realeukin9 (IL9) mRNA expression. Method: The specific primers were designed according to the conserved sequence of human IL9 gene. Total RNAs were extracted from human peripheral blood mononuclear cells (PBMC) which were stimulated by PMA and ionomycin, then the RNAs were transcribed reversely into cDNAs. The plasmid standards were constructed. The sensitivity and specificity of the method were evaluated. The relative expression levels of human IL9 mRNA in PBMCs from patients with Graves′ disease (GD) and healthy controls were detected by this method. Results: The coefficient of correlation of the standard curve of this method was 0.995-0.998, melting curve analysis showed single peak. The relative expression levels of human IL9 mRNA in GD group were higher than that in healthy control group (0.01). Conclusion: The method to detect IL9 by SYBR GreenⅠbased qRTPCR was good in sensitivity, specificity and linear function. It can be used as a standard method of qRTPCR for detection of human IL9 expression. 　　[Key words] interleukin 　　早先认为IL是由Th2细胞分泌的一种细胞因子，在抗寄生虫感染及过敏性哮喘中发挥重要作用。近期研究发现有一种新的CD4+T细胞亚群，主要分泌IL及IL，称之为“Th9细胞”, 在炎症反应及自身免疫性疾病中发挥重要的作用[1-3]。我们建立了一种检测人IL表达的实时荧光定量PCR(realquantitative PCR, qRTPCR)方法，并应用于Graves病患者外周血单个核细胞(peripheral blood mononuclear cell, PBMC)中人IL的检测，为进一步研究IL在临床中的应用提供了实验手段。 　　1 材料和方法 　　1.1 材 料 　　1.1.1 研究对象 Graves病患者组22例(男5例，女17例)，平均年龄(31.4±9.4)岁，均为经江苏大学附属人