小鼠CD4＋CD25＋T调节细胞的分离、纯化及其功能检测

Isolation, purification and function of mouse CD4 ＋ CD25＋ regulatory T lymphocytes in vitro

目的 探讨小鼠CD4＋CD25＋T调节细胞（Treg）的分离培养、纯化及其部分功能检测.方法 采用免疫磁珠分离法（MACS）对分离小鼠的脾淋巴细胞进行分选CD4＋CD25＋Treg细胞,锥虫蓝细胞染色检测其活性,流式细胞仪检测分选所得活性细胞的纯度,酶联免疫吸附试验（ELISA）检测培养上清液中白细胞介素（IL）-2、IL-10水平的浓度.结果 MACS分离的CD4＋CD25＋Treg细胞的纯度达83%～96%.体外培养中Treg组、T组和混合组IL-2和IL-10的平均水平分别为：（10.25±2.31）、（40.32±8.05）ng/L;（5 8.21±13.05）、（11.52±3.01）ng/L;（39.54±12.82）、（31.25±4.36）ng/L,数据差异有统计学意义（P＜0.05,P＜0.01）.结论 采用MACS系统两步法,可获得高纯度、具有免疫抑制功能的Treg细胞,该细胞对CD4＋CD25-T细胞的免疫抑制作用可能是通过IL-10对IL-2的调节作用实现的.

Objective To establish a method for isolation and purification of CD4＋ CD25＋ regulatory T lymphocytes （Treg）, and to identify partial functions of these cells. Methods Lymphocytes were isolated from the mouse spleens and then CD4＋ CD25＋ T cells were sorted by magnetic bead cell sorting （MACS） system. The purity of CD4＋ CD25＋ T cells were analyzed by flow cytometry （FCM）. The activity of them was detected by trypan blue staining. Interleukin （IL）-2 and IL-10 levels in culture supernatant were determined by enzyme linked immunosorbent assay （ELISA）. Results The purity of CD4＋ CD25＋ T cells sorted by MACS was 83%-96%. There was significant difference in the levels of IL-2 and IL-10 secreted by lymphocytes among CD4＋ CD25＋ Treg group [ （10.25±3.31）, （40.32±8.05） ng/L], CD4＋ CD25- T group [（58.21±13.05）,（11.52±3.01）ng/L] and mixed culture group [ （39.54±13.82）, （31.25±4.36）ng/L （P〈0.05,P〈0.01）.Conclusion High purity of CD4＋ CD25＋ Treg with immune regulatory function can be isolated by MACS. The immunosuppression of CD4＋ CD25＋ Treg to CD4＋ CD25- T was related to the regulation of IL-10 through IL-2.