| 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TCR BV 基因家族设计上游引物, 共同的TCR β链恒定区(BC) 基因设计下游引物, 荧光定量PCR扩增26个TCR BV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TCR β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TCR β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TCR BV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。 |
| 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 |
| DOI： |
| 投稿时间：2013-04-07修订日期：2013-06-16 |
| 基金项目: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). |
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| Gene melting curve spectratyping technique in analyzing polymorphism of T-cell receptor beta chain variable region in CD4+ T cells of tuberculosis patients |
| ZHANG Dong-mei1,SUN Xiao-lei1*,DUAN Yi-nong1,TANG Wei2,ZHOU Ming-ming1,LI Zheng-xin1,ZHANG Ju1,ZHANG Jie1 |
(1. Departmet of Pathogen Biology, College of Medicine, Nantong University, Nantong 226019, Jiangsu, China
2. Department of Infectious Diseases, the Affiliated Hospital of Nantong University, Nantong 226019, Jiangsu, China
*Corresponding author.) |
| Abstract: |
| 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. |
| Key words: CD4-positive T-lymphocytes T cell receptor beta genes genetic polymorphism pulmonary tuberculosis |
