实时定量PCR评价小鼠胸功能方法的建立.docVIP

实时定量PCR评价小鼠胸腺功能方法的建立 作者：张华华；曾耀英；何贤辉；刑飞跃　　(暨南大学组织移植与免疫实验中心，广东??广州??510632) 摘要：目的??建立实时定量PCR检测小鼠胸腺及脾脏T淋巴细胞中信号结合T细胞受体删除环(sjTRECs)水平的方法，推测其中的初始T细胞的数目，评价胸腺功能，从而为胸腺功能研究及胸腺研究模型的建立提供研究依据和方法。方法 提取小鼠胸腺和脾脏淋巴细胞基因组DNA，PCR扩增目的片段。纯化构建标准质粒的RAG2片段，构建标准重组质粒并鉴定。优化PCR体系后，实时定量PCR反应建立标准曲线,并检测不同品系(BALB/c和C57BL/6)成年小鼠胸腺及脾脏淋巴细胞sjTRECs含量。结果 成功构建标准质粒。确定最适实时定量PCR反应条件后，在实时定量PCR中建立可信度高的标准曲线。建立的方法分析表明，这两个不同品系的sjTRECs含量统计学差异不显著。结论 成功建立定量检测小鼠T淋巴细胞中sjTRECs含量从而推测其中的初始T细胞数目的方法，为胸腺功能研究及胸腺研究模型的建立提供研究依据和方法。 关键词：实时定量PCR；T细胞受体删除环；幼稚T细胞 中图分类号：Q93??文献标识码：A??文章编号：1673-4254(2006)01-0062-04 Real-time quantitative PCR for evaluating murine thymic functionZHANG Hua-hua；ZENG Yao-yin；HE Xian-hui；XING Fei-yueInstitute of Tissue Transplantation and Immunology, Ji’nan University, Guangzhou 510632, ChinaAbstract: Objective To establish a real-time quantitative PCR method for detecting the levels of the signal joint T cell receptor excision circles (sjTRECs) in murine thymocytes and spleen lymphocytes for determining the amount of nave T cells and evaluating the thymic function. Methods The genomic DNA was extracted from murine thymocytes and splenocytes for PCR amplification of the target fragments. After purification of the PCR product, the recombination-activating gene 2 (RAG2) fragment was cloned into pGEMT-Easy vector to construct the standard plasmid. After PCR optimization, the standard curve was obtained and the samples (thymocytes and splenocytes of BALB/c and C57BL/6 mice) were detected for sjTRECs by real-time quantitative PCR. Results The standard plasmid was correctly constructed, and the standard curve with high reliability was obtained. No statistical difference was observed in sjTREC contents in the T lymphocytes between the two mouse strains. Conclusions Real-time quantitative PCR for sjTREC analysis is established successfully, which offers an important means for thymic function analysis and a reliable model establishment for study the thymus.Key words: real-time polymerase chain reaction; T