小鼠CD4＋ CD25＋免疫调节性T细胞体外扩增及功能检测的研究

Amplification of mice CD4＋ CD25＋ regulatory T cells in vitro and it＇s function detection

目的 探讨小鼠CD4＋ CD25＋调节性T细胞（Tregs）的体外扩增方法及扩增后Tregs的免疫功能.方法 利用免疫磁珠法分离小鼠Tregs;采用anti-CD3mAb＋rIL-2和Allo-APC＋rIL-2两种方法进行体外扩增,通过TRANSWELL培养系统、细胞因子表达及CFSE染色标记等方法检测Tregs免疫抑制作用的机制.结果 体外分离的Tregs纯度为89.5％;活细胞率约为98％.Tregs扩增的倍数在两组各时间点差异无统计学意义.扩增后CD4＋ CD25＋T细胞、CD4＋T细胞和CD4＋ CD25-T细胞的每分钟计数（CPM）分别为1 470、12 700和14 300.混合淋巴细胞反应（MLR）中当CD4＋：CD25＋T细胞的比例为1∶1时,抑制率为79％,比例为8∶1时,抑制率为33％.CFSE标记后,进入分裂周期的CD4＋T细胞的百分比在未加入CD4＋ CD25＋T细胞组为83.7％,加入CD4＋ CD25＋T细胞组为31.7％,差异具有统计学意义（P=0.0006）.CD4＋ CD25＋T细胞对CD4＋T细胞的增殖抑制率在Transwell和Non-Transwell的培养系统中分别为＜5％和95％.Transwell培养组中IL-2含量高于Non-Transwell组,差异具有统计学意义[Transwell培养组为（158.33±2.08）pg/mL,Non-Transwell组为（23.00±2.00） pg/mL,P ＜0.0001].结论 采用免疫磁珠法分离小鼠Tregs可行,Tregs可有效扩增;Tregs的作用机制为抑制CD4＋T细胞的增殖及抑制CD4＋T细胞分泌IL-2;其抑制作用需要细胞-细胞间接触;体外扩增后的Tregs仍具有免疫特性,其体外免疫抑制作用增强.

Objective To detect the immune function of CD4 ＋ CD25 ＋T cells （Tregs）,amplified in vitro.Methods We used the micro-magnetic beads to isolate Tregs.Two means were adopted to amplify Tregs （anti-CD3mAb group and allo-APC group）.The function of Tregs was detected with the Transwell system,cytokine detection and CFSE marker.Results The purity of Tregs is 89.5％.The amplification times were not significantly deferent between the two groups.The counts per minute（CPM） of CD4 ＋ CD25 ＋T cell,CD4 ＋ T cell and CD4 ＋ CD25T cell is 1 470,12 700 and 14 300 respectived.In mixed lymphocyte reaction （MLR）,when the ratio of CD4/CD25＋ T cell is 1∶ 1 and 8∶1,suppression ratio is 79％ and 33％.We labeled CD4＋T cells with CFSE,the percent is 83.7 ％ without Tregs,and 31.7 ％ with Tregs （P =0.0006）.The proliferation inhibition ratio inducedby is 5％ in Transwell system and 95％ in Non-Transwell system.The concentration of IL-2 in Transwell system is significantly higher than that in Non-Transwell（158.33 ± 2.08） pg/mL vs （23.00 ± 2.00） pg/mL,P ＜ 0.0001）.Conclusion The method of micro-magnetic beads can be used to isolate Tregs.Tregs can be amplified effectively and inhibit CD4 ＋T cells proliferation.Tregs amplified in vitro still have immune with function with enhanced immune-suppressive effect.