基于免疫磁珠分选的调节性T细胞亚群嵌合性定量分析

为了探讨免疫分选偶联短串重复序列（STR）方法用于调节性T细胞（Treg）嵌合性定量监测的可行性,以淋巴细胞不同比例混合制备14组人工嵌合体血样,以单个核细胞为起点,以免疫磁珠阴性和阳性选择法获取CD4＋CD25＋Treg细胞,分选后的Treg细胞再进行16位点的STR长度多态性分析。结果显示,从分选后的Treg细胞提取的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Treg细胞嵌合性定量分析方法,对造血嵌合体的检出灵敏度和准确性在1%-10%,可用于造血干细胞移植术后嵌合体监测。

Quantitative Chimerism Analysis of Regulatory T Cell Subsets Based on Immunomagnetic Sorting

The aim of study was to explore the feasibility of quantitative chimerism analysis of regulatory T （Treg） cells using immune sorting coupling short tandem repeat （STR） method. 14 sets of artificial chimera samples were prepared by mixed lymphocytes according to different proportion. The CD4＋CD25＋ Treg cells were harvested by negative and positive selection of immunomagnetic beads, then the STR polymorphisms of 16 loci in sorted Treg cells was analyzed. The results showed that the DNA amount extracted from sorted Treg cells was fit for STR detection. All STR alleles specific for recipient or donor could be detected and the quantitative results were consistent with theoretic values in over 10% recipient chimeras. But only partial recipient alleles could be detected and the quantitative results were different from theoretic values in less then 1% recipient chimeras. It is concluded that a quantitative chimerism analysis of Treg cell based on immune sorting is established. The sensitivity and accuracy for chimera detection are 1% to 10%, and this method can be used to monitoring hematopoietic chimerism following allageneic hematopoietic stem cell transplantation.