自身免疫病患者外周血中B细胞激活因子基因表达水平的测定

Measurement of the B-lymphocyte stimulator gene expression on peripheral blood mononuclear cells from patients with autoimmune disease

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

Objective To establish a specific fluorogenic quantitative RT-PCR (FQ-RT-PCR) method for detecting the expression of B-lymphocyte stimulator (Blys) gene in peripheral blood mononuclear cells (PBMCs) and investigate the relationship between BlyS mRNA expression and autoimmune diseases. Method A real-time quantitative reverse transcription (RT)-PCR was set up,based on fluorescent TaqMan methodology,to examine the specific gene expression of Blys in 19 autoimmune disease patients,20 subclinical patients 20 healthy subjects,and 8 other diseased patients with high serum levels of immunoglobulins.Results In 19 autoimmune disease patients,the BlyS mRNA expression ranged from 9.7×10 5 to 3.2×10 8 copy/μg RNA,the mean value was (8.4±7.9)×10 7 copy/μg RNA.In 20 subclinical patients the BlyS mRNA expression level was (1.3±1.2)×10 6 copy/μg RNA;and for 20 healthy subjects the BlyS mRNA expression level was (1.7±1.4)×10 5 copy/μg RNA.And the BlyS mRNA expression level in 8 other diseased patients with high serum immunoglobulins was (1.2±1.2)×10 6 copy/μg RNA.Conclusion BlyS gene expression detected with FQ-PCR had an accurate and speedy result.The BlyS mRNA expression level was significantly elevated in autoimmune diseases,with the production of autoantibodies and higher serum IgG.And the BlyS mRNA might be used as a useful marker of early diagnosis of autoimmune diseases.