【摘要】 目的:建立检测人T细胞TCRBD基因(Diversity Gene)经历重排的连接介导PCR方法(Ligation Mediate dPCR,LMPCR),为T细胞重排和疾病的关系研究提供基础。方法:在人TCRβ链BD1与BD25′端重组信号序列(RSS)前,BD1与BD23′端RSS后各设计两套用于巢式PCR引物;设计并合成和双链RSS平断裂末端相接的特殊接头(BWLinker),提取胸腺组织、正常人和急性T淋巴细胞白血病(TALL)外周血单个核细胞(PBMC)样本总DNA,和BWlinker连接,进行巢式PCR,PCR产物琼脂糖电泳分析,阳性产物做胶回收,并克隆测序鉴定。结果:在1例胸腺组织提取的总DNA中证实存在BD1和BD25′和3′的RSS断裂末端,在2例TALLs的PBMC中检测到BD25′和3′的RSS断裂末端,2例正常人PBMC中未能检测到RSS断裂末端,阳性的LMPCR产物通过测序鉴定,和基因组中序列完全相符合。结论:1例胸腺组织和2例TALL的PBMC中检测到RSS断裂末端,提示TCRBD基因正经历重排,测序结果表明建立的监测TCR重排的LMPCR方法可靠,可用于T细胞重排模型和相关疾病的机制研究。更多还原

【Abstract】 Objective:To established a special ligation-mediated PCR for analyzing the breaks end of recombination signal sequence of human TCR BD gene.Methods:Designing four primers in front of nanomer-spacer(12)-heptamer(RSS 9-12-7) and four primers behind of heptamer-spacer(23)-nanomer(RSS 7-23-9) from human TCR diversity gene and synthesizing A partially double-stranded oligonucleotide linker(BW-linker).Isolating total DNA from thymus,and from the PBMC samples of T-lineage acute lymphoblastic leukemia(T-ALLs) and healthy controls,linking BW-linker to the end of DNA RSS and identified the signal ends by PCR and sequencing analysis.Results:In the thymus(one samples) could find RSS breaks end of TCR BD1 and TCR BD2,and in PBMC of two T-ALL patients could find RSS breaks end of TCR BD2,but in two controls,it is negative.The products of LM-PCR has exactly nucleotide sequence corresponding to the human TCR BD gene.Conclusion:The positive LM-PCR results demonstrated ongoing TCR gene recombination in thymus and T-ALL patients PBMC,the LM-PCR represents a simple and reliable tool for detecting human TCRβ chain gene rearrangement.更多还原