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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。
Objective To observe the expression and chemotactic characteristics of ConA-activated regulatory T cell surface chemokine receptors in vitro and provide clues for the use of regulatory T cells to induce immune tolerance. Methods Normal peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers. Immunomagnetic beads were used to sort CD4 + T cells. FITC-An-tiCD4 antibody, APC-AntiCD25 antibody and PE-AntiCD127 antibody were used to separate CD4i CD127loCD25hi-int cell. Purified regulatory T cells and CD4 + CD25-T were stimulated with ConA (10μg / mL) for 0, 24 and 48h, respectively. Chemokines CCL1, CCL5, CCL20 and CCL22 were used for chemotaxis assays. Chemotactic properties of lower regulatory T cells and CD4 + CD25-T cells. Meanwhile, the expression of CCR4 and CCR6 was detected by flow cytometry. Results The purity of regulatory T cells isolated was 97.4% with a viable cell ratio of 95% and a yield of 4.1%. CCL1, CCL20 and CCL22 all chemotactic regulatory T cells, and chemotactic efficiency changes with time after ConA activation. The chemotactic index of CCL1 and CCL22 on regulatory T cells was significantly higher than that of CD4 + CD25-T cells; CCL20 had high chemotactic index on regulatory T cells and CD4 + CD25-T cells; Sex was significantly weaker than CD4 + CD25-T cells. After ConA stimulation, the expression of regulatory T cell chemokine receptors CCR4 and CCR6 were significantly higher than that of control cells (CD4 + CD25-T cells). With the prolongation of ConA stimulation, the expression of CCR4 in both groups of cells continued to increase. The expression of CCR6 in regulatory T cells was significantly increased 24h after stimulation, and the expression of CCR6 decreased slightly after 48h. The expression of CCR6 in control cells also showed a similar trend. Conclusion 1) Negative magnetic separation combined with flow cytometry technology can sort out the higher purity and viability of regulatory T cells. 2) Regulatory T cells have different chemotactic properties compared to CD4 + CD25-T cells. CCL1 chemotactic effect on regulatory T cells more specific, CCL22 chemotaxis stronger CCL5 chemotaxis is weak, while CCL20 on CD4 CD25 T cells and regulatory T cell chemotaxis are strong. 3) The chemotactic effects of chemokines CCL1, CCL20 and CCL22 on regulatory T cells within 48 h after ConA stimulation increased with stimulation time. 4) ConA stimulation can enhance the receptor CCR4, CCR6 expression. Suggesting that the expression of chemokine receptors and cell activation state, and different receptors expression trends.