【摘要】 目的:建立实时荧光定量PCR(RFQPCR)检测人B细胞激活因子受体TACImRNA含量的方法,初步探讨健康献血员外周血单个核细胞(PBMC)中TACImRNA表达水平。方法:在TACI基因高保守区设计特异性的引物和荧光探针,PCR扩增目的片段并实时检测产物的荧光强度,根据标准品建立的标准曲线,由软件自动计算出待测样本中TACImRNA准确含量,并以TACImRNA和β2MmRNA含量的比值作为评价TACI表达水平的指标。结果:本法检测TACImRNA含量的线性范围为109～101pg/ml,批内和批间重复性测定的CV分别为2.97%～9.32%和5.86%～10.29%。40例健康献血员样本的PBMC中35例(87.5%)检出有TACImRNA表达,范围为0.02～2.11。结论:成功建立RFQPCR检测TACImRNA含量的方法,具有较好的检测灵敏度和重复性,为进一步探讨TACImRNA表达水平与自身免疫性疾病发病机制之间的关系奠定了基础。更多还原

【Abstract】 Objective:To establish real-time fluorescence quantitative polymerase chain reaction (RFQ-PCR) for measurement of the expression level of B lymphocyte stimulator receptor- TACI mRNA in peripheral blood mononuclear cell(PBMC)in 40 healthy donors. Methods:Specific primers and TaqMan probe have been designed , and fluorescence of the PCR product was detected continuously during amplification. According to the standard curve created by plasmid DNA, the expression level of TACI in clinical samples has been determined using software, and the results were presented as the ratios of TACI mRNA to β2-microgluobulin(β2M) mRNA.Results:The detection range of the assay was from 10~9 to 10~1 pg/ml, the coefficient of variation values for both intra-experimental and inter-experimental reproducibility ranged from 2.97% to 9.32% and 5.86% to 10.29%, respectively. In 40 PBMC samples of healthy donors, 87.5% expressed TACI mRNA, and the value ranged from 0.02 to 2.11.Conclusion:This assay had high sensitivity and reproducibility, and suitable for clinical practice, which will certainly play a significant role in approaching the mechanism of autoimmune diseases.更多还原