人外周血CD4^+CD25^+Foxp3^+调节性T细胞分选及扩增方法的建立

Isolation and amplification of CD4^+CD25^+Foxp3^+Treg cells from human peripheral blood

目的建立人外周血CD4^+CD25^+Foxp3^+调节性T细胞(Treg)的免疫磁珠分选法(MACS)及体外扩增方法 ,并对扩增前后Treg细胞纯度及表型进行鉴定。方法流式细胞术检测人外周血Treg细胞占CD4^+T细胞的比例;免疫磁珠法分选CD4^+CD25^+Treg细胞,用CD3/CD28单抗包被的Dynalbeads联合IL2共同刺激CD4^+CD25^+Treg细胞进行体外扩增培养,在扩增培养的第0、7、14、21天分别进行细胞计数及检测细胞的活性,取扩增培养第0天及第14天细胞行流式细胞术检测扩增前后细胞纯度、主要表面标记及Foxp3的表达情况。结果正常人外周血CD4^+CD25^+Foxp3^+Treg水平占CD4^+T细胞(7.94±1.86)%,MACS能够成功分选出CD4^+CD25^+Foxp3^+Treg细胞,分选平均纯度达(95.86±0.65)%,细胞活性>95%;以CD3/CD28单抗包被的Dynalbeads联合IL2共同刺激CD4^+CD25^+Treg细胞进行外扩增培养后,细胞有明显扩增。经2周扩增后,细胞扩增倍数达到原细胞数量的(21.33±1.53)倍,扩增后细胞Foxp3的表达由(95.86±0.65)%下降至(93.71±1.30)%,但差异无统计学意义(P>0.05)。结论免疫磁珠分选法能够分选出高纯度的CD4^+CD25^+Foxp3^+调节性T细胞,体外成功扩增CD4^+CD25^+Foxp3^+调节性T细胞,扩增前后Treg细胞的纯度及表型无明显变化。

Objective This research was designed to investigate an effective method （magnetic activated cell sorting, MACS） to isolate and amplify regulatory T cells （Treg） in vitro from peripheral blood of healthy donors and to identify the purity of these cells. Methods Peripheral blood was obtained from healthy donors. The proportions of CD4^＋CD25^＋Foxp3^＋ Treg cells in CD4^＋T cells were analyzed by flow cytometry. CD4^＋ CD25^＋Foxp3^＋Treg cells were purified by MACS and expanded in vitro cultures in addition of CD3/CD8 Dynalbeads and IL2. The cells number and activity after expanding the cells for 0 day, 7 days, 14 days and 21 days were determined. The purity of cells the main surface marker and the level of Foxp3 were detected by flow cytomctry. Results The proportions of CD4＋CD25~Foxp3＋ Regulatory T Cells （Treg） in CD4^＋T cells was （7.94±1.86）%. Treg cells could be successfully isolated by the MACS and the average classifying purity could reach （95.86±0.65）% and the activity of classified cell could he higher than 95%. The amplification achieved （21.33±1.53） times after cultures in addition of CD3/CD8 Dynalbeads and IL2 for 2 weeks in vitro. After amplification, the expression of Foxp3 decreased from （95.86±0.65）% to （93.71 ±1.30）%. No significant differences were observed （P〉O.05）. Conclusion It is concluded that CD4^＋CD25^＋Foxp3^＋Treg cells can be successfully isolated by the MACS. The amplification of Treg ceils is successful in vitro. Expression of surface markers and Foxp3 does not obviously change after amplification.