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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 T cell receptor (TCR) β chain variable region (BV) gene in peripheral blood CD4 + T cells of patients with pulmonary tuberculosis by fluorescence quantitative PCR and gene melting curve spectratyping (GMCS) Sex. Methods Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) from 15 healthy people and 30 pulmonary tuberculosis patients. The total RNA was reverse transcribed into cDNAs. The upstream primers were designed from 26 human TCRBV gene families, and the common TCRβ chain constant Region (BC) genes were designed as downstream primers, and 26 TCRBV gene families were amplified by fluorescence quantitative PCR. The lineage of the samples was analyzed by DNA melting curve in real-time quantitative PCR. Results The melting curves of PCR products of the same BV locus were the same in TCR β chain of 15 healthy human peripheral blood T cells. However, compared with healthy controls, the melting curve profiles of 26 BV sites in TCRβ chain of peripheral blood of 30 patients with tuberculosis differed from 6.7% to 33.3% (P <0.05, P <0.01) . In addition, 13 out of 26 loci had melting curve spectral differences of ≥20%. Conclusion Fluorescent quantitative PCR combined with GMCS analysis of TCRBV gene family pedigree, the method is stable and simple, and can better monitor the TCR β-chain profile of T cells in peripheral blood of patients with pulmonary tuberculosis.