实时定量PCR检测自身免疫中APR IL mRNA表达

Detection of the proliferation-inducing ligand APRIL mRNA in autoim-mune disease using real-time quantitative PCR assay

建立检测增殖诱导配体(a proliferation-inducing ligand,APRIL)mRNA的SYBR GreenⅠ实时定量PCR方法,了解自身免疫性疾病[系统性红斑狼疮(SLE)、类风湿性关节炎(RA)]患者外周血单个核细胞(PBMC)中APRIL的表达水平,研究APRIL mRNA的表达与自身免疫性疾病发病机制、预后之间的关系。方法构建克隆载体pGEM-T Easy-APRIL作为定量模板,Line Gene FQD-33A荧光定量PCR仪定量检测58例自身免疫性疾病(SLE、RA)确诊病人和20例正常健康献血者的外周血APRIL mRNA表达含量,以APRIL mRNA和内参GAPDH mRNA含量比值作为APRIL表达水平的指标。结果58例自身免疫病患者的APRIL mRNA表达范围3.95～192,均值为29.68±4.5。20例正常对照标本PBMC APRIL mRNA的表达范围3.1～18.7,均值为10.56±2.0。自身免疫病患者外周血中APRIL mRNA的表达水平高于正常对照组,其中久治不愈或疗效差者APRIL mRNA表达水平明显高于初发患者及治疗后缓解患者,但后两组差异无显著性意义。结论成功建立了人APRIL基因表达含量的SYBR GreenⅠ实时定量检测方法。自身免疫性疾病(SLE、RA)患者的APRIL mRNA的表达含量升高,其中疗效差久治不愈患者有更高表达水平。研究APRIL表达和自身免疫病发展、早期诊断、预后等相关性,为寻找自身免疫病治疗新靶点打下基础。

To establish a SYBR Green 1 real-time quantitative PCR assay to detect the expression of APRIL gene in peripheral bloods of patients with autoimmune diseases, such as systemic lupus erythematosus（SLE）and rheumatoid arthritis（RA）, so as to make certain of the relationship between the expression of APRIL mRNA and the pathogenesis as well as the prognosis of autoimmune diseases, plasmid PGEM-T-easy-APRIL was cloned and was used as the standard quantitative template. The SYBR Green I real-time quantitative PCR assay was set up to detect the expression of APRIL mRNA in peripheral bloods of 58 patients with autoimmune diseases and 20 normal controls using line gene FQD-33A fluorescence quantitative PCR amplificator, and the ratio of APRIL mRNA with that of the reference mRNA of glyceraldehydle-3-phosphate dehydrogenase（GAPDH） was used as the indices of the expression level. It was demonstrated that the expression ranges of APRIL mRNA of 58 patients with autoimmune diseases and 20 normal controls were 3.95-192 （mean value： 29.68 - 5.4） and 3.1-18.7（mean value： 10.56 - 2.0）respectively, indicating a higher expression level of APRIL mRNA in patients with autoimmune diseases than that of normal controls. In addition, the APRIL mRNA expression level of patients without response after long period of treatment or with poor response to therapy was even higher than that of patients discovered recently or patients with good response to therapy. However there was no significant difference in the expression levels among these cases. It is evident that the SYBR Green I real-time quantitative PCR assay was successfully established for use to detect the expression of APRIL gene in peripheral bloods of patients with autoimmune diseases, and this method of assay was proved to be accurate and reliable, thus providing the basis for the studies on the pathogenesis, early diagnosis, prognosis and searching for the novel therapeutic target for autoimmune diseases.