NOD小鼠未成熟树突状细胞体外诱导淋巴细胞生成并扩增调节性T细胞的研究

Research on induction and expansion of regulatory T cells by immature dendritic cells of NOD mice in vitro

目的探索非肥胖糖尿病(non-obese diabetic,NOD)小鼠未成熟树突状细胞(immature dendritic cell,imDC)诱导并扩增调节性T细胞(regulatory Tcells,Treg)的优化方案。方法分离、培养NOD小鼠骨髓来源的imDC和淋巴细胞,并进行混合淋巴细胞培养,分别加入白介素(IL)-2、转化生长因子(transforming growth factor,TGF)-β1、抗CD3抗体和抗CD28抗体刺激淋巴细胞增殖、分化。实验分为4组:对照组(淋巴细胞+IL-2)、实验1组(淋巴细胞+imDC+TGF-β1+IL-2)、实验2组(淋巴细胞+imDC+抗CD3抗体+抗CD28抗体+IL-2)、实验3组(淋巴细胞+imDC+抗CD3抗体+抗CD28抗体+TGF-β1+IL-2)。用流式细胞仪检测各组培养3d后淋巴细胞的扩增情况及混合培养6d淋巴细胞中CD4、CD25及Foxp3的表达。结果 imDC和淋巴细胞混合培养3d后,对照组、实验1组、实验2组和实验3组增殖指数分别为(1.04±0.03)%、(1.26±0.02)%、(1.36±0.01)%和(1.55±0.02)%。实验3组的增殖指数较实验1组和实验2组明显升高(均为P<0.01)。混合培养6d后,对照组、实验1组、实验2组和实验3组的CD4+CD25+T淋巴细胞亚群所占比例分别为(2.41±0.02)%、(17.98±1.02)%、(30.67±2.68)%、(42.84±1.41)%,而CD4+CD25+T淋巴细胞中Foxp3+细胞的比例4组分别为(65.55±1.41)%、(86±1.72)%、(70.29±1.39)%、(92.3±1.99)%,实验3组的淋巴细胞CD4+CD25+、Foxp3+的表达水平较实验1组和实验2组明显升高(均为P<0.01)。结论在imDC和淋巴细胞混合培养时加入TGF-β1、抗CD3抗体、抗CD28抗体可明显刺激imDC诱导扩增Treg,是体外诱导扩增Treg的理想方案之一。

Objective To explore a novel protocol of efficient induction and expansion of CD4＋CD25＋Foxp3＋regulatory T cells with immature dendritic cells（imDC）from non-obese diabetic（NOD）mouse in vitro.Methods The imDC and lymph cells from NOD mouse were isolated,mixed and then co-cultured.Interleukin-2（IL-2）,transforming growth factor（TGF）-β1,CD3 antibody（anti-CD3）as well as CD28 antibody（anti-CD28）were added to the mixed lymphocyte reaction（MLR）to induce regulatory T cells respectively.The experiment were divided into 4 groups：control group（lymph cells＋IL-2）,experimental group 1（lymph cells＋imDC＋TGF-β1＋IL-2）,experimental group 2（lymph cells＋imDC＋anti-CD3＋anti-CD28＋IL-2）and experimental group 3（lymph cells＋imDC＋anti-CD3＋anti-CD28＋TGF-β1＋IL-2）.MLR were cultured for 3 days and the lymphocyte expansion was detected by flow cytometry.The expressions of CD4,CD25 and Foxp3 on lymphocytes were also detected by flow cytomer on day 6 after co-culture.Results Proliferation index（PI）of control group,experimental group 1,experimental group 2 and experimental group 3 were（1.04±0.03）%,（1.26±0.02）%,（1.36±0.01）% and（1.55±0.02）% respectively.The PI of experimental group 3 was significantly higher than those of experimental group 2 and experimental group 1（all in P0.01）.The proportion of CD4＋CD25＋T cells in control group,experimental group 1,experimental group 2,experimental group 3 were（2.41±0.02）%,（17.98±1.02）%,（30.67±2.68）%,and（42.84±1.41）% respectively.The proportion of Foxp3＋cells in CD4＋CD25＋T cells of control group,experimental group 1,experimental group 2,experimental group 3 were（65.55±1.41）%,（86.00±1.72）%,（70.29±1.39）% and（92.30±1.99）% respectively.The expressions of CD4＋CD25＋and Foxp3＋in experimental group 3 were significantly higher compared with experimental group 2 and experimental group 1（all in P0.01）.Conclusion In the co-culture of imDC and lymphocytes,TGF-β1,anti-CD3 and anti-CD28 can significantly induce imDC and subsequently lead to regulatory T cells expansion,which is an efficient method to induce and expand regulatory T cells.