传染性单核细胞增多症患儿B细胞刺激因子mRNA表达水平研究

Measurement of B-lymphocyte stimulator mRNA expression in children with infectious mononucleosis by real-time fluorenscence quantitative method

目的B淋巴细胞增殖是传染性单核细胞增多症免疫变化的始发环节,该实验采用实时荧光定量PCR(RFQ-PCR)技术检测儿童传染性单核细胞增多症患儿外周血人B细胞刺激因子(BLyS)mRNA表达水平,为临床诊断该病提供新的实验依据。方法在BLyS基因的高保守区设计相应引物和荧光探针,实时检测PCR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靶基因中BLyS的水平。结果RFQ-PCR检测靶基因mRNA含量的线性范围为109ng/L^101ng/L,批内和批间重复性测定的变异系数v分别为1.88%~5.89%和6.32%~12.34%。18例传染性单核细胞增多症患儿样本的BLySmRNA水平的表达水平均显著高于正常儿童(P<0.01)。结论成功建立RFQ-PCR检测BLySmRNA含量的方法,具有较好的检测灵敏度和重复性;而且发现BLyS在儿童传染性单核细胞增多症发病过程中可能起了重要作用,BLyS可以作为儿童传染性单核细胞增多症的一个参考指标。

Objective B cell multiplication plays a key role in infections mononucleosis, The present study was designed to detect the expression of B-lymphocyte stimulator （BLyS） mRNA in peripheral blood using real-time fluorescence quantitative polymerase chain reaction （RFQ-PCR） in children with infectious mononucleosis in order to explore the role of BLys in this disorder. Methods Specific primers and TaqMan probes of BLyS were designed, and fluorescence of the PCR products were detected continuously during amplification, According to the standard curves created by plasmid DNA, the expression level of target genes in clinical samples were calculated using Stata Software version 8.0, and the results were presented as the ratio of copies of target gene mRNA to 62 microglobulin （β2M） mRNA copies. BLyS mRNA expression in peripheral blood was measured by RFQ-PCR in 18 children with infectious mononucleosis and the results were compared with those measured in 15 healthy controls. Results The range of target gene mRNA detected by REQ-PCR was from 10^9ng/L to 10^1ng/L, The coefficient of variation for intra-experimental and inter-experimental reproducibility ranged from 1.88% to 5.89% and 6.32% to 12.34%, respectively, BLyS mRNA expression in peripheral blood in children with infectious mononucleosis were significantly higher than that in controls （1.65±0.10 vs 0.56±0.08 ; P 〈 0.01）. Conclusions RFQ-PCR has a high sensitivity and reproducibility for the measurement of BLyS mRNA expression. BLyS may be involved in the development of infectious mononucleosis.