一种动态检测小鼠微量血中T细胞受体重排删除环含量的方法

目的:通过检测比较外周血(颈动脉大量采血和微量隐静脉采血)、胸腺组织和脾脏组织等不同成分或组织中T细胞受体重排删除环(T cell receptor rearrangement excision circles,TREC)的含量,并建立一种通过微量外周血PCR预扩增的方法,评估胸腺输出功能。方法:采用C57BL/6小鼠作为实验对象,分成幼年组(5周龄,n=10)和成年组(15周龄,n=10)。经过隐静脉采血及颈动脉采血后处死小鼠,取小鼠胸腺器官和脾脏器官,提取基因组DNA(g DNA),隐静脉微量血g DNA通过PCR预扩增,提纯后再和颈动脉血、胸腺组织和脾脏组织g DNA一起进行实时定量PCR,分析各组织成分TREC的表达水平及差别。结果:幼年组胸腺组织及外周血中TREC的含量比成年组高,且二者趋势基本一致;而脾脏组织结果与其相反;幼年组小鼠胸腺组织中TREC的含量比脾脏组织中高,成年组小鼠结果与之相反;微量外周血经过预扩增后再进行实时定量PCR的结果与直接定量PCR结果一致,且更高效。结论:研究提供了一种新型高效的动态检测T细胞受体重排删除环和检测胸腺输出功能的方法。

Establishment ofA Novel Method of Dynamically Detection T Cell Receptor Rearrangement Excision Circles Content frommicro-volume Mouse Blood

Objective:To detect the content of T cell receptor rearrangement excision circles (TREC) in peripheral blood(carotid artery blood and trace saphenous vein blood), thymus and spleen tissue, and to compare the differences among them, we establish a practical method of evaluatingthymic output function by real-time PCR coupling with pre-PCR amplification of the raw micro blood genomic DNA.Methods:C57BL/6 mice were randomized into young group (5 weeks, n=10) and adult group (15 weeks, n=10). Blood were sampled from saphenous vein dynamically or carotid artery when mice were sacrificed, and while thymus and spleen were collected. All the above samples were extracted of genomic DNA and then analyzed TREC levels by qPCR, except for the trace saphenous vein blood samples, which was pre-PCR amplification before qPCR.Results:TREC levels in thymus tissue and peripheral blood from the young group are higher than those from adult group, in contrast with the content of spleen tissue among different groups, thymic TREC was higher than splenic ones in young group, which was opposite to the trend in adult group, prePCR amplification coupling with qPCR from trace saphenous vein blood samples could achieve the similar TREC content compared with direct qPCR from peripheral blood, which was more efficient.Conclusion:Data from our study provides a new and efficient dynamic method to detect TREC and explore the thymus output function in a longitude study.