艾滋病毒感染者外周血T细胞TCR β链CDR3谱系漂移的监测
Detection of CDR3 skewing of TCR beta gene repertoire in peripheral blood of HIV infected people
-
摘要: 目的 利用荧光定量PCR溶解曲线分析技术监测HIV感染者外周血T细胞受体(TCR) 链 CDR3 谱系漂移(单/寡/多克隆增生)。 方法 提取6例HIV感染者、4名正常人外周血单个核细胞(peripheral blood mononuclear cell-PBMC)中的总核糖核酸(RNA),反转录成互补脱氧核糖核酸(cDNA),设计人26个TRBV 基因家族上、下游引物,荧光定量PCR(FQ-PCR)扩增26个TRBV 基因各家族CDR3谱系,溶解曲线法分析各家族CDR3谱系的单/寡/多克隆增生。 结果 正常人外周血T细胞TCR 链26个家族CDR3表达频率不一致,各家族PCR产物的溶解曲线谱型图显示多克隆增生的高斯分布,呈现溶点不同的CDR3多态性,6例HIV感染者的外周血TCR 链CDR3 谱系的26个家族CDR3表达频率不一致,患者各家族PCR产物的溶解曲线谱型图上,多数家族为多克隆增生的高斯分布,但每个患者均出现数量不等的单克隆和寡克隆增生家族。 结论 荧光定量PCR溶解曲线法监测人TCR 链CDR3谱系漂移的方法稳定简便,可以用来监测正常人和HIV感染者外周血T细胞TCR 链CDR3 谱系漂移。Abstract: Objective To detect the CDR3 skewing of TCR beta gene repertoire in the peripheral blood of HIV infected people by using real-time fluorescence quantitative reverse transcription polymerase chain reaction (FQ-PCR) with DNA melting curve analysis. Methods The total RNA of peripheral blood mononuclear cell (PBMC) from 4 healthy donors and 6 HIV infected people were transcribed reversely into cDNA. The cDNA of 26 TRBV gene families CDR3 was amplified by FQ-PCR, The monoclonal/oligoclonal/polyclonal CDR3 spectratyping were analyzed with DNA melting curve. Results The 26 TRBV families CDR3 showed different frequency in healthy donors and patients. The CDR3 spectratyping of 26 TRBV families showed polyclonal peak (Gaussian distribution) in healthy donors but showed different monoclonal/oligoclonal/polyclonal peak in HIV infected people with DNA melting curve analysis. Conclusion The technique of FQ-PCR with DNA melting curve analysis can be used for detecting the CDR3 skewing of TCR beta gene repertoire in HIV infected people.
下载: