一种检测小鼠T淋巴细胞中sjTRECs含量简易方法的研究

A simple method for the measurement of sjTRECs levels in murine T lymphocytes

目的 建立一种快速、简单的检测小鼠胸腺和脾脏淋巴细胞的信号结合T细胞受体删除环(signaljointTCRrearrangementexcisioncircles,sjTRECs)水平的方法,并研究其准确性,以期推测初始T细胞含量从而评价胸腺功能。方法 根据标准质粒荧光定量PCR的扩增曲线选择合适有效的扩增循环次数;优化PCR条件;梯度稀释的标准质粒(10 3 - 10 8拷贝)PCR扩增后使用图像分析软件包分析琼脂糖凝胶电泳的图像,测量PCR产物带的光密度,经统计获得标准曲线及方程。将样本的PCR产物带的光密度代入标准曲线方程,从而获得样本的sjTRECs水平读数,并和实时定量PCR结果比较。结果 获得适合的PCR条件;成功建立可信度高的标准曲线;与实时定量PCR的检测结果比较,小鼠sjTRECs含量随年龄变化的趋势一致。结论 成功建立研究方法,与实时定量PCR评价sjTRECs水平得到明显相似的结果。

Objective To study a simple and rapid method and it's veracity to quantify signal joint TCR rearrangement excision circles (sjTRECs) levels of murine thymocytes and spleen lymphocytes.From the result,to evaluate the amount of nave T cell and the thymus function.Methods Choose correct and effective PCR cycle number according to standard plasmid's amplification curve in real-time PCR;PCR optimization;a image analysis software was used to measure the brightness of PCR product bands on the agarose gel.The standard curve could be obtained and the sjTRECs levels in samples could be read out by comparison with standard curve.The result was compared with the result of real-time PCR.Results Effective PCR condition was found;the highly believable standard curve was successfully constructed;with ageing of murine,the content of sjTRECs had the same tendency as real-time PCR.Conclusion The method is developed successfully.The remarkable similarity between the two methods indicates that the gel scanning method is an effective and reliable for analysis of sjTRECs level.