“荧光定量PCR溶解曲线法”监测人TCRβ链CDR3谱系漂移技术的建立

Establishment of the real-time fluorescence quantitative reverse transcription polymerase chain reaction with DNA melting curve analysis for detecting the CDR3 skewing of TCR beta gene repertoire in the human peripheral blood

目的建立"荧光定量PCR溶解曲线分析技术"监测人外周血T细胞TCRβ链CDR3谱系漂移(单/寡/多克隆增生)。方法提取4例正常人、9例大肠癌患者外周血单个核细胞(peripheral blood mononuclear cell-PBMC)中的总RNA,逆转录成cDNA,以26个人TRBV基因家族设计上游引物,共同的TRBC基因设计下游引物,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TCRβ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"(melting curve spectratyping)呈现溶点不同的CDR3多态性,为多克隆增生的高斯分布;9例大肠癌患者的外周血TCRβ链CDR3谱系的26个家族CDR3表达频率不一致,有的患者部分家族呈缺失状态,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溶解曲线分析TCR CDR3谱系漂移技术",方法稳定简便,能较好的监测正常人和临床样本外周血T细胞TCRβ链CDR3谱系漂移(单/寡/多克隆增生)。

Objective To establish the technique of the real-time fluorescence quantitative reverse transcription polymerase chain reaction(FQ-PCR) with DNA melting curve analysis for detecting the CDR3 skewing of TCR beta gene repertoire in the human peripheral blood.Methods The total RNA of peripheral blood mononuclear cell(PBMC) from 4 healthy donors and 9 colorectal cancer patients were transcripted reversely into cDNA.The cDNA of 26 TRBV gene family CDR3 was amplified by the FQ-PCR;the monoclonal/oligoclonal/polyc...