目的建立实时定量PCR检测小鼠T淋巴细胞中信号结合T细胞受体删除环(sjTREC)水平和T细胞受体β链可变区(TRBV)CDR3的方法,推测其中的初始T细胞的数目和克隆性,评价胸腺输出功能及免疫重建,从而为胸腺输出功能及移植后T细胞免疫提供可靠的研究方法。方法分别提取小鼠脾脏细胞基因组DNA和总RNA。PCR方法扩增DNA标本目的片段。纯化鉴定并构建含sjTREC片段的质粒标准品。建立稳定的实时定量PCR反应检测小鼠脾脏淋巴细胞sjTREC含量的方法。采用半巢式PCR扩增TRBV基因产物,采用ABI3700分析TRBV谱型。结果构建了可靠的sjTREC和TCRα质粒标准品。在实时定量PCR中建立了可靠的标准曲线。建立的定量PCR方法分析可成功用于脾细胞的sjTREC含量的检测。8周龄BALB/c小鼠脾细胞sjTREC含量为(626±115)拷贝/105个脾细胞。结论建立了可靠的定量检测小鼠T淋巴细胞中sjTREC含量的方法,建立了分析T细胞谱型的方法,为研究生理和病理条件下胸腺输出功能及T细胞免疫重建改变提供了可靠的方法。 Objective To establish a real-time quantitative PCR method to detect the level of signal-bound T cell receptor-deleted loop (sjTREC) and CDR3 of T cell receptor β chain variable region (TRBV) in mouse T lymphocytes, and to estimate the number of initial T cells Clonality, evaluation of thymus output function and immune reconstruction, so as to provide a reliable research method for thymus output function and post-transplantation T cell immunity. Methods Genomic DNA and total RNA of mouse spleen cells were extracted respectively. PCR method to amplify the target DNA fragment. Purification Identification and construction of plasmid standards containing sjTREC fragments. To establish a stable real-time quantitative PCR method to detect the spleen lymphocyte sjTREC content in mice. TRBV gene products were amplified by semi-nested PCR and TRBV patterns were analyzed by ABI3700. Results The reliable sjTREC and TCRα plasmid standards were constructed. The establishment of a reliable standard curve in real-time quantitative PCR. The established quantitative PCR method can be successfully used to detect the content of sjTREC in spleen cells. The sjTREC content of 8-week-old BALB / c mice spleen cells was (626 ± 115) copies / 105 splenocytes. Conclusion A reliable and quantitative method for quantitative determination of sjTREC in mouse T lymphocytes was established and a method for the analysis of T cell profiling was established. This method provided a reliable method for studying thymus export function and T cell immune reconstitution under physiological and pathological conditions.