基因熔解曲线谱型图技术分析肺结核患者CD4^+T细胞受体β链可变区基因多态性  

Gene melting curve spectratyping technique in analyzing polymorphism of T-cell receptor beta chain variable region in CD4^+T cells of tuberculosis patients

作　　者：张冬梅[1] 孙晓雷[1] 段义农[1] 汤伟[2] 周鸣鸣[1] 李正欣[1] 张菊[1] 张洁[1] 

机构地区：[1]南通大学医学院病原生物学教研室,南通226019 [2]南通大学附属医院传染科,南通226019

出　　处：《第二军医大学学报》2013年第12期1317-1322,共6页Academic Journal of Second Military Medical University

基　　金：南通市科技计划项目(S2008049;BK2013027);江苏省高等学校大学生实践创新训练计划(201313993012X)~~

摘　　要：目的利用荧光定量PCR结合基因熔解曲线谱型图(gene melting curve spectratyping,GMCS)技术分析肺结核病患者外周血CD4+T细胞中T细胞受体(TCR)β链可变区(BV)基因多态性。方法提取15例健康人和30例肺结核患者外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中的总RNA,反转录成cDNA,以26个人TCRBV基因家族设计上游引物,共同的TCRβ链恒定区(BC)基因设计下游引物,荧光定量PCR扩增26个TCRBV基因家族谱系,样品谱系用荧光定量PCR中的DNA熔解曲线进行分析。结果在15例健康人外周血T细胞TCRβ链中,同一BV位点PCR产物的熔解曲线谱型相同。但是与健康人相比,30例肺结核患者的外周血TCRβ链26个BV位点熔解曲线谱型存在差异,差异比率为6.7%~33.3%,差异具有显著性(P<0.05,P<0.01)。此外,26个位点中有13个位点熔解曲线谱型差异比率≥20%。结论荧光定量PCR结合GMCS技术分析TCRBV基因家族谱系情况,方法稳定、简便,能较好地监测肺结核患者外周血T细胞TCR的β链谱型。Objective To analyze the polymorphism of the T-cell receptor （TCR） beta chain variable region（BV） gene family spectratyping of peripheral blood CD4＋T cells in tuberculosis patients by gene melting curve spectratyping （GMCS） technique combined 24 h fluorescence quantitative （FQ）-PCR. Methods The total RNA of peripheral blood mononuclear cells （PBMCs） from 15 healthy blood donors and 30 tuberculosis patients were used to obtain the cDNA. The cDNAs were amplified by FQ-PCR using 26 different forward primers and the same reverse primer, and the spectratype was analyzed by DNA melting curve. Results The DNA melting curves of each TCR BV family in 15 healthy donors showed the same spectratype. However, the DNA melting curves of 26 TCR BV families showed different spectratypes in 30 tuberculosis patients compared with healthy blood donors, and the different rate was from 6.7% to 33.3%. The result indicated that the difference rate of 26 TCR BV families in tuberculosis patients was significantly different from that of healthy blood donors （P〈0.05, P〈0.01）. In addition, the different rates of 13 TCR BV families （totally 26 families） in tuberculosis patients were more than 20%. Conclusion This study suggests that FQ-PCR combined with GMCS is a convenient and stable method for detecting the TCR beta gene repertoire in the peripheral blood of tuberculosis patients.

关 键 词：CD4阳性T淋巴细胞 T细胞受体β基因 遗传多态现象 肺结核 

分 类 号：R392.22[医药卫生—免疫学]