摘要
目的建立人外周血单个核细胞来源的CD4+CD25+调节性T细胞体外扩增培养方法 ,研究体外扩增后细胞功能改变。方法 Ficoll-Paque密度梯度分离健康人外周血单个核细胞,免疫磁珠纯化CD4+T细胞,流式分选CD4+CD25+调节性T细胞。用抗人CD3/CD28单抗和IL-2联合刺激CD4+CD25+调节性T细胞,检测其Foxp3、IL-10和TGF-β表达改变。结果人外周血单个核细胞分离纯化得到纯度98%的CD4+CD25+调节性T细胞,Foxp3表达率为95%;使用IL-2加抗CD3/CD28单抗刺激6周后细胞数量可扩增1 000倍;扩增后细胞Foxp3的表达和IL-10、TGF-β的分泌均显著降低。结论本研究成功建立了高纯度大量CD4+CD25+调节性T细胞纯化和体外扩增方法,研究了CD4+CD25+T细胞体外扩增后功能表型的改变。
In this study, we aim to establish an in vitro expansion method for CD4＋CD25＋ regulatory T cells （Treg） and detect its function. CD4＋CD25＋reg was separated and purified from peripheral blood mononuclear cell by flow cytometry with 98% purity and 95% of purified cells were Foxp3 positive. The purified CD4＋CD25＋ Freg was further stimulated in vitro with IL-2 and anti-CD3/CD28 monoclonal antibody for 1 to 6 weeks. Cell proliferation of stimulated Treg was detected by radioactive H3 incorporation assay and a thousandfold increase was found after 6- week cuhure. Furthermore, the expression of Foxp3 and the secretion of IL-10/TGF-β were found significantly lower in stimulated Tregs than those in non-stimulated. In conclusion, this study successfully established an expansion method for CD4＋CD25＋ Tregs, and Treg lose their Foxp3 marker after in vitro expansion.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2014年第6期504-507,共4页
Immunological Journal
