Objective: To establish a method for amplification of mouse CD4 + CD25 + Foxp3 + regulatory T cell in vitro. Methods :CD4+T cells were derived from C57BL/6 mouse spleen,selected by Mini MACS,and cultured in 24 wells plate for 3 weeks,the plate was coated with aCD3 monoclonal antibody,the medium system included aCD28 monoclonal antibody,rhIL-2 and Rapamycin with RPMI1640 medium and 15% FBS,under the 371 ,5%CO2 and saturated humidity incubator. After 3 weeks,CD4 + CD25 + T cells were detected by FCM, the expression of Foxp3 mRNA was measured with real-time PCR, MLR and proliferation inhibition were used to exam the function of CD4 + CD25 + T cells, IL-10 and TGF-(31 of supernatant were measured by ELISA. Results: CD4 + CD25 + T cells were derived from CD4 + T cells,it was ( 76. 05 ± 2. 73 )% , higher than the group without Rapamycin( 52. 17 ± 1. 36 )% ( P < 0. 001 ), the Foxp3 mRNA of CD4 + CD25 + T cells in Rapamycin group was 5 folds higher than the group without Rapamycin ( P <0. 001 ),the ability of proliferation was 0. 29 folds higher than the group without Rapamycin( P < 0. 001 ), the ability of inhibition to CD4 + T cells was 3. 6 folds higher than the group without Rapamycin( P <0.001 ),the IL-10 of supernatant was 1.8 folds higher and TGF-β1 of supernatant was 1.6 folds higher than the group without Rapamycin( P < 0.001 ). Conclusion: Mouse CD4 + CD25 + Foxp3 + Tregs were successfully expanded with aCD3,aCD28,rhIL-2 and Rapamycin in vitro.%目的:本研究旨在探讨CD4+CD25+Foxp3+调节性T细胞体外扩增的方法.方法:采用磁珠分选小鼠CD4+T细胞,αCD3单克隆抗体包被24孔板,加入αCD28单克隆抗体、雷帕霉素、rhIL-2,培养3周后,流式细胞仪测定培养细胞中CD4+CD25+T细胞的含量,实时定量PCR检测CD4+CD25+T细胞Foxp3 mRNA的表达;单向混合淋巴细胞反应和增殖抑制试验测定扩增的CD4+CD25+T细胞的增殖及其抑制功能;ELISA检测培养上清中IL-10和TGF-β1的含量.结果:小鼠CD4+T细胞培养3周后,CD4+CD25+T细胞达(76.05±2.73)%,高于未加雷帕霉素组(52.17±1.36)%(P＜0.001),磁珠分选的CD4+CD25+T细胞Foxp3 mRNA的表达是未加雷帕霉素组的5倍(P＜0.001),增殖能力是未加雷帕霉素组的0.29倍(P＜0.001),对CD4+T细胞增殖抑制能力是未加雷帕霉素组的3.6倍(P＜0.001),培养上清中IL-10和TGF-β1分别是对照组的1.8倍和1.6倍(P＜0.001).结论:小鼠CD4+T细胞在含有1 μg/ml的αCD28、rhIL-2 100 U/ml和终浓度为10 nmol/L雷帕霉素的培养体系中培养3周后能有效扩增CD4+CD25+Foxp3+调节性T细胞.
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