【摘要】 目的体外动态观察ConA激活的调节性T细胞表面趋化因子受体的表达变化及其趋化特性,为利用调节性T细胞诱导免疫耐受提供线索。方法常规分离正常健康人外周血单个核细胞,免疫磁珠阴性分选CD4+T细胞;加FITC-An-tiCD4抗体,APC-AntiCD25抗体,PE-AntiCD127抗体上流式细胞仪分选出CD4hiCD127loCD25hi-int细胞。纯化的调节性T细胞与CD4+CD25-T分别用ConA(10μg/mL)刺激0、24、48h后,用趋化因子CCL1、CCL5、CCL20、CCL22做趋化实验,观察各趋化因子作用下调节性T细胞与CD4+CD25-T细胞的趋化特性。同时,流式细胞仪检测CCR4与CCR6的表达。结果分离得到的调节性T细胞纯度为97.4%,活细胞率为95%,得率:4.1%。CCL1、CCL20、CCL22均可趋化调节性T细胞,且在ConA激活后趋化效率随时间而改变。CCL1与CCL22对调节性T细胞的趋化指数显著高于CD4+CD25-T细胞;CCL20对调节性T细胞和CD4+CD25-T细胞趋化指数都很高;CCL5对调节性T细胞趋化性则显著弱于CD4+CD25-T细胞。ConA刺激后,调节性T细胞趋化因子受体CCR4、CCR6的表达均明显高于对照组细胞(CD4+CD25-T细胞)。随着ConA刺激时间延长,两组细胞CCR4的表达均持续增强;调节性T细胞CCR6的表达在刺激24h后表达明显增强,48h后CCR6的表达略有减弱,呈下降趋势。对照组细胞CCR6的表达也呈现相似的趋势。结论1)磁珠阴性分选结合流式细胞仪技术可分选出较高纯度及活率的调节性T细胞。2)调节性T细胞与CD4+CD25-T细胞相比,二者具有不同的趋化特性。CCL1对调节性T细胞的趋化作用较特异,CCL22趋化作用较强,CCL5趋化作用较弱.而CCL20对CD4+CD25-T细胞和调节性T细胞趋化作用都强。3)ConA刺激后48h内趋化因子CCL1,CCL20,CCL22对调节性T细胞的趋化作用随刺激时间而增强。4)ConA刺激可以增强受体CCR4、CCR6表达。提示趋化因子受体的表达与细胞活化状态有关,且不同受体表达变化趋势不同。更多还原

【Abstract】 Objective To explore the chemotaxis and chemokine receptors expression of conA-activated CD4+CD25+ regulatory T cells in vitro,for benefit the study of regulatory T cell-induced immune tolerance.Methods The peripheral blood mononuclear cells were separated using Ficoll,and then the CD4+ cells were enriched negatively by immunomagnetic beads.FITC-AntiCD4,APC-AntiCD25,PE-AntiCD127 were added into CD4+ cells,then CD4hiCD127loCD25hi-int T cell was sorted by flow cytometry and identified as regulatory T cells.Purified regulatory T cells and CD4+CD25-T cells were induced by ConA(10 μg/mL)for 0,24,and 48 h,respectively.The chemotactic effects of chemokines CCL1,CCL5,CCL20,and CCL22 on regulatory T cells and CD4+CD25-T cells were observed.After stimulation with ConA(10 μg/mL),the purified regulatory T cells and CD4+CD25-T cells were labelled with PE-Cy7-anti CCR4 and PE-anti CCR6 for observing the expression changes of CCR4,and CCR6 by flow cytometry.Results The purity,survival rate,and yield of sorted regulatory T cells was 97.4%,95%,and 4.1%,respectively.CCL1,CCL20,and CCL22 all had the chemotactic effects on regulatory T cells,furthermore after the cells were activated,the chemotactic efficacy changed as time goes on.The chemotactic indexes of CCL1 and CCL22 on regulatory T cells were higher than that on CD4+ CD25-T cells;the chemotactic index of CCL20 on regulatory T cells and CD4+CD25-T cells was high;CCL5 had a small chemotactic effect on regulatory T cells.After stimulated by ConA,the expression of CCR4 and CCR6 on regulatory T cells were higher than that on CD4+CD25-cells.Along with the constitutively ConA stimulation,the expression of CCR4 on both CD4+CD25+ T cell group and control group were increased continuously.The expression of CCR6 on CD4+ CD25+ T cells increased evidently at 24 h but decreased at 48 h after ConA stimulation,while the expression of CCR6 on control group had same trend.Conclusion The chemotactic effects of CCL1 on regulatory T cells are specific,while chemotactic effect of CCL22 on regulatory are strong;CCL20 had a higher chemotactic effect on regulatory T cells and CD4+CD25-T cells.ConA activation could enhance the chemotactic effects of CCL1,CCL20,and CCL22 on regulatory T cells in vitro and also can increase the expression levels of CCR4 and CCR6.The expression of chemokine receptors are correlated to continuous time of stimulation,and different receptor has different trends.更多还原