摘要
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1μg/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。
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 αCD3 monoclonal antibody,the medium system included αCD28 monoclonal antibody,rhIL-2 and Rapamycin with RPMI1640 medium and 15% FBS,under the 37℃,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-β1 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）%（P0.001）,the Foxp3 mRNA of CD4＋CD25＋T cells in Rapamycin group was 5 folds higher than the group without Rapamycin（P0.001）,the ability of proliferation was 0.29 folds higher than the group without Rapamycin（P0.001）,the ability of inhibition to CD4＋T cells was 3.6 folds higher than the group without Rapamycin（P0.001）,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（P0.001）.Conclusion：Mouse CD4＋ CD25＋ Foxp3＋Tregs were successfully expanded with αCD3,αCD28,rhIL-2 and Rapamycin in vitro.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2012年第2期109-113,共5页
Chinese Journal of Immunology
