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目的 建立测定B细胞激活因子 (B lymphocytestimulator ,BlyS)mRNA含量的荧光定量反转录 聚合酶链反应 (RT PCR)法 ,并用来检测自身免疫病 [系统性红斑狼疮 (SLE)、类风湿关节炎 (RA) ]患者外周血单个核细胞 (PBMCs)中BlyS的基因表达水平 ,探讨BlyS基因表达水平与自身免疫性疾病发病机制的关系。方法 构建克隆载体 pMD18 T BlyS作为定量模板 ,基于TaqMan荧光探针技术 ,建立实时荧光RT PCR方法在GeneAmp 5 70 0型检测仪上定量检测 19例自身免疫病(SLE、RA)确诊病人、2 0例亚临床病人 (主要是抗核抗体阳性 )、8例其他对照性疾病患者 (自身抗体阴性 ,免疫球蛋白升高 )、2 0名正常健康献血者的外周血BlySmRNA表达含量。结果 19例自身免疫病确诊病人的PBMCs中均有BlySmRNA的表达 ,范围从 9 7× 10 5～ 3 2× 10 8拷贝 /μgRNA ,均值为 (8 4± 7 9)× 10 7拷贝 /μgRNA ;2 0例亚临床病人的强度为 8 6× 10 4～ 3 8× 10 6拷贝 /μgRNA ,均值为 (1 3± 1 2 )× 10 6拷贝 /μgRNA ;2 0名正常健康人的强度为 5 5× 10 4～ 4 9× 10 5拷贝 /μgRNA ,均值为 (1 7± 1 4 )× 10 5拷贝 /μgRNA ;8例自身抗体阴性而免疫球蛋白升高的其他疾病患者的强度为 5 8× 10 5～ 3 5× 10 7拷贝 /μgRNA ,均值为 (1 2±
Objective To establish a reverse transcription polymerase chain reaction (RT PCR) method for the determination of B lymphocyte stimulator (BlyS) mRNA and to detect autoimmune diseases [systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) patients with peripheral blood mononuclear cells (PBMCs) BlyS gene expression levels, to explore the BlyS gene expression levels and the pathogenesis of autoimmune diseases. Methods The cloning vector pMD18 T BlyS was constructed as a quantitative template. Based on the TaqMan fluorescent probe technique, real-time fluorescence RT PCR was used to detect 19 cases of autoimmune diseases (SLE, RA) on the GeneAmp 5 70 0 detector. Cases of sub-clinical patients (mainly anti-nuclear antibody positive), 8 patients with other control diseases (autoantibodies negative, elevated immunoglobulin), 20 normal healthy blood donors BlySmRNA expression levels. Results The expression of BlyS mRNA in PBMCs from 19 patients with autoimmune diseases was detected. The average value of BlyS mRNA was from 9 7 × 10 5 to 3 2 × 10 8 copies / μg RNA with an average of (84 ± 7 9) × 10 7 copies / μg RNA. The intensity of 20 sub-clinical patients was 8 6 × 10 4 ~ 3 8 × 10 6 copies / μg RNA with an average of (13 ± 1 2) × 10 6 copies / μg RNA. The intensity of 20 normal healthy subjects was 5 5 × 10 4 ~ 4 × 10 5 copies / μg RNA, with an average of (17 ± 1 4) × 10 5 copies / μg RNA. The intensity of other 8 patients with autoantibodies negative and immunoglobulin increased was 58 × 10 5 ~ 3 5 × 10 7 copies / μg RNA, mean (1 2 ±