摘要
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.
出处
《国际免疫学杂志》
CAS
2014年第6期526-530,共5页
International Journal of Immunology
基金
吉林省科技厅资助项目(201215201)