FQ-PCR溶解曲线技术分析白血病患者PBMC中T细胞TCRα/βCDR3谱系特征  

Analysis of TCR CDR3 spectratyping of αβ T cells in peripheral blood mononuclear cells with leukemia by using FQ- PCR DNA melting curve technique

作　　者：孙永苹[1,2] 石彬[1] 朱红倩[3] 汤贤英[1] 马锐[1] 姚新生[1] 

机构地区：[1]遵义医学院免疫学教研室,贵州遵义563099 [2]遵义医学院第二临床学院,广东珠海519100 [3]贵州省人民医院血液内科,贵州贵阳550002

出　　处：《遵义医学院学报》2014年第1期39-47,共9页Journal of Zunyi Medical University

基　　金：国家973前期专项(NO:2008CB517310);贵州省自然科学基金项目(NO:2007-2122);国际合作计划基金项目(NO:[2008]700105)

摘　　要：目的采用FQ-PCR溶解曲线技术分析临床白血病患者外周血单个核细胞(PBMC)中T细胞TRAV和TRBV基因各家族的CDR3谱系特征。方法提取4例健康志愿者和15例临床白血病患者PBMC中mRNA,逆转录成cDNA,FQ-PCR扩增32个TRAV基因和24个TRBV基因家族的CDR3区,FQ-PCR溶解曲线技术分析CDR3谱系特征,并测序分析单克隆家族CDR3区氨基酸序列的组成。结果 4例健康志愿者PBMC中α/βT细胞的CDR3谱系FQ-PCR溶解曲线峰型图,均表现为多峰图;而15例白血病患者PBMC中α/βT细胞CDR3谱系FQ-PCR溶解曲线峰型图,多数患者出现寡或偏峰型图、低或无峰型图、典型的单峰型图;不同白血病患者异常峰的频率不一致;单峰型图TRAV和TRBV PCR产物基因测序,未发现不同患者存在完全相同的CDR3区。结论FQ-PCR溶解曲线技术能很好的分析白血病患者PBMC中TCRα/βCDR3谱系的单、寡、多克隆性增殖;非T细胞白血病患者PBMC中TCRα/βCDR3的单峰(或寡峰)家族T细胞,可能来源于机体对肿瘤应答的T细胞,而在T细胞白血病患者,可能为肿瘤T细胞或机体抗肿瘤T细胞。本实验可为进一步研究白血病的免疫应答机制和个体化治疗提供基础。Objective To analyze the CDR3 sequence of TRAV and TRBV gene repertoire in Peripheral Blood Mononuclear Cells （PBMC） with leukemia by using real - time fluorescence quantitative reverse transcription polymerase chain reaction （ FQ - PCR） with DNA melting curve analysis. Methods Total RNA from PBMC of fifteen patients with leukemia and four healthy donors were isolated and reverse tran- scripted into cDNA. The cDNA of 32 TRAV and 26 TRBV gene families were amplified by FQ -PCR. The features of 34 TRAV and 26 TRBV gene families were determined using DNA melting curve analysis technique. According to the melting curve, we selected the PCR products of TRAV and TRBV familiesto show monoclonal expansion. In addition, the CDR3 spectratyping of the monoclonal families were an- alyzed. Results The melting curves of TCR CDR3 spectratyping of α/β T Cells in PBMC showed poly- clonal in the four health donors. However, most patients with leukemia showed different oligo -clonal, skew peak, low peak, no peak and typical single peak. Additionally, different patients have their own rate of abnormal peak, and the gene sequence of CDR3 region of monoelonal expansion of TRAV or TR- BV families were different. Conclusion Current technique of FQ - PCR with DNA melting curve analysis technique is commendable for detecting the clonal of CDR3 spectrum. Furthermore the single peak （or oligopeak） families ofα/β CDR3 of TCR in PBMC with non T cell leukemia probably is derived from a tumor response of T cells. However, in the case of T cell leukemia, that may be derived from tumor T cells or antitumor T cells. To sum up, present data would possibly provide indication to the study of the mechanism of immune response and individualized immunotherapy of leukemia.

关 键 词：白血病 TCR CDR3 FQ-PCR 溶解曲线 

分 类 号：R733.7[医药卫生—肿瘤]