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目的:建立基于免疫分选的T淋巴细胞亚群嵌合性定量分析的方法。方法:以淋巴细胞不同比例混合制备14组人工嵌合体血样,以免疫磁珠阳性选择法获取CD4+和CD8+T细胞亚群,分选后的细胞再进行16位点的短串重复序列(STR)多态性分析。结果:分选后T细胞亚群提取的DNA量能满足STR检测要求,当嵌合体中受体比例≥10%时,16个STR位点均能检出受体和供体特异性等位基因,按照STR峰面积计算的受体比例与理论值相符;当受体比例≤1%时,仅有部分位点能检出受体特异性等位基因,实测嵌合率与理论值存在差异。结论:建立了基于免疫分选的T细胞亚群嵌合性定量分析方法,可用于造血干细胞移植术后嵌合体监测。
OBJECTIVE: To establish a method for the quantitative analysis of chimerism in T lymphocyte subsets based on immune sorting. Methods: Fourteen groups of artificial chimeric blood samples were prepared by mixing different proportions of lymphocytes. CD4 + and CD8 + T cell subpopulations were obtained by immunomagnetic beads positive selection method. The sorted cells were further analyzed by a short tandem repeat (STR ) Polymorphism analysis. Results: The amount of T-cell subsets extracted after sorting could meet the requirement of STR detection. When the proportion of receptors in the chimera was ≥10%, all 16 STR loci could detect the receptor and donor-specific alleles , And the proportion of recipients calculated according to the area of STR peak is consistent with the theoretical value. Only when the proportion of receptors is less than 1%, only some of the loci can detect receptor-specific alleles, and the measured chimeric rates are different from the theoretical values. Conclusion: The method of quantitative analysis of T cell subsets chimerism based on immune sorting is established and can be used for the monitoring of chimerism after hematopoietic stem cell transplantation.