摘要
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:用 Primer Express v2.0软件,根据GenBank提供的序列,在人IL-2Rα mRNA的第2和第3外显子之间设计一对引物和一条TaqMan探针。提取总RNA,加入已优化的一步法FQ-RT-PCR反应体系,扩增产物经凝胶电泳后切胶回收,并与pUCm-T载体连接。制备好的重组质粒转化感受态细胞进行克隆。经测序分析确定为特异性片段后,用T7RNA聚合酶转录为cRNA,作为FQ-RT-PCR系列浓度标准品。评价FQ-RT-PCR检测 IL-2Rα mRNA的特异性、线性、精密度和探针稳定性等。定量测定HLA-B27阳性活动组与阴性非活动组强直性脊柱炎患者外周血单个核细胞IL-2Rα mRNA基因的表达水平,结合血浆可溶性白细胞介素-2受体 (sIL-2R)浓度,探讨与炎症活动的相互关系。结果:成功构建了cRNA标准品。该方法的线性范围为7～107 cells/ml,其批内CV8.4%,批间CV9.6%;扩增产物克隆的双向测序结果经BLAST比较、分析,证实为IL- 2Rα mRNA特异性片段。对各30例HLA-B27阳性与阴性的强直性脊柱炎的测定表明:与正常人对照组结果比较,HLA-B27阳性组与HLA-B27阴性组IL-2Rα mRNA和sIL-2R都有明显增加(P<0.01)。IL-2Rα mRNA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- 2Rα mRNA与sIL-2R比较更能反映强直性脊柱炎患者的免疫状态,可能为免疫药物的疗效评价和药物筛选提供基因表达水平上的信息。
Objective:To construct and evaluate real-time fluorescence quantitative poiymerase chain reaction for detecting IL-2Ra mRNA in ankylosing sondylitis(AS) on TaqMan technique. Methods: A pair of primers and a probe with MGB were designed by Primer EXPress V2. O software program . Using the peripheral blood monocytes (PBMC) of homo sapiens as samples , total RNA was isolated from fresh PBMC . RNA was amplified by the real-time FQ-RT-PCR. The specificy of the recombined plasmid was tested by agarose gel eletrophoresis and sequencing analysis . IL-2Rα recombined plasmid was transcribed to cRNA by T7 RNA poiymerase for Serial Standard materials of FQ-RT-PCR. A new method was created to quantify IL-2Rα mRNA in ideal condition sensitivity, reproducibity and efficiency of the FQ-RT-PCR was evaluated and used ,combined with sIL-2R,for clinical application of AS. Results:Minimum limit of FQ-RT-PCR system was 7cells/ml and linear range from (7-107)cells/ml . The intra-and-inter-assay cofficient variation(CV) was 8. 4% and 9. 6% respectively. Recombined plasmid contained the target fragment was specificity and accuracy by BLAST. Standard reference was constructed successfully. RT-PCR product in AS with HLA-B27 positive groups were higher than HLA-B27 negtive groupsand health controls (P <0. 01), HLA-B27 negtive groups and HLA-B27 positive groups were not different (P >0. 05) . The sensitivity of IL-2Rα mRNA was 96. 7%. Conciusions:Real Time FQ-RT-PCR of JL-2Rα mRNA is constructed successfully. This is easy, rapid and more sensitive , accurate and reliable method for quantifing IL-2Rα mRNA. There is highly statistical significance,compared with sIL-2R ,on the expression of IL-2Rα mRNA and inflammatory states between AS and control group and effective information for adminstration of patients.
