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目的利用TaqMan实时荧光定量逆转录聚合酶链反应(FQ-RT-PCR)检测强直性脊柱炎(AS)患者外周血单个核细胞白细胞介素2受体α(IL-2Rα)mRNA的表达水平,并进行疾病的活动性相关分析。方法根据GenBank提供的序列设计一对引物和一条TaqMan探针。提取外周血单个核细胞(PBMC)总RNA,加入FQ-RT-PCR反应体系,产物切胶回收与pUCm-T载体连接,用T7RNA聚合酶转成cRNA,制备系列浓度参照物。对其特异性、线性、精密度和探针稳定性进行评价。定量HLA-B27阳性组与阴性组AS患者PBMC的IL-2RαmRNA基因表达并探讨与血浆可溶性白细胞介素2受体(sIL-2R)的相互关系。结果成功构建了IL-2RαmRNA的cRNA参照物。线性范围7~107cells/ml,批内CV8·4%,批间CV9·6%。对各30例HLA-B27阳性与阴性AS的IL-2RαmRNA检测表明,与正常对照组结果比较,阳性组与阴性组IL-2RαmRNA和sIL-2R有明显差异(P<0·01)。IL-2RαmRNA对炎症活动评价的灵敏度为96·7%。结论FQ-RT-PCR具有灵敏、特异、结果重复性好等优点;IL-2RαmRNA比sIL-2R更能反映强直性脊柱炎患者的炎症活动程度。
Objective To detect the expression of interleukin 2 receptor alpha (IL-2Rα) mRNA in peripheral blood mononuclear cells in patients with ankylosing spondylitis (AS) by TaqMan real-time fluorescence quantitative reverse transcription polymerase chain reaction (FQ- And the disease activity correlation analysis. Methods According to the sequences provided by GenBank, a pair of primers and a TaqMan probe were designed. Total RNA of peripheral blood mononuclear cells (PBMCs) was extracted and added into FQ-RT-PCR reaction system. The product was gel-excised and ligated into pUCm-T vector. T7 RNA polymerase was used to transform cRNA to prepare concentration reference. Its specificity, linearity, precision and probe stability were evaluated. The mRNA expression of IL-2Rα in peripheral blood mononuclear cells from patients with HLA-B27 positive and negative AS was quantified and its relationship with plasma soluble interleukin-2 receptor (sIL-2R) was explored. Results The cRNA reference of IL-2Rα mRNA was successfully constructed. Linear range of 7 ~ 107cells / ml, intra-CV8.4%, inter-CV9.6%. The detection of IL-2RαmRNA in 30 cases of HLA-B27 positive and negative AS showed that the IL-2RαmRNA and sIL-2R were significantly different between the positive group and the negative group (P <0.01) compared with the normal control group. The sensitivity of IL-2Rα mRNA to the assessment of inflammatory activity was 96.7%. Conclusion FQ-RT-PCR is sensitive, specific and reproducible with good results. IL-2Rα mRNA reflects the inflammatory activity of patients with ankylosing spondylitis better than sIL-2R.