趋化因子CCL11-糖胺聚糖-趋化因子受体CCR3的相互作用研究

目的 探究趋化因子CCL11与受体CCR3、糖胺聚糖（glycosaminoglycans,GAGs）相互作用过程及机制,为深入阐明CCL11-GAGs-CCR3相互作用关系提供理论参考。方法 利用基因工程技术,构建筛选了CCR3-EGFP单分子表达水平的CHO稳转细胞系,利用全内反射荧光成像（total internal reflection fluorescence,TIRF）与等温滴定量热（isothermal titration calorimetry,ITC）技术研究了不同体外溶液条件下GAGs与CCL11的相互作用,并利用趋化实验及活细胞单分子成像实验考察了GAGs-CCL11对CCR3-EGFP稳转细胞趋化行为的调控及CCR3-EGFP在细胞膜上聚集状态的影响。结果 随着硫酸软骨素链长度的增加,其与CCL11结合放热增多,表明其相互作用力增强,其促进CCL11聚集作用增强。单分子荧光成像技术结合趋化试验研究发现,不同种类及不同比例的GAGs均会影响CCL11与CCR3的相互作用,GAGs的加入,抑制了CCL11对CCR3-EGFP稳转细胞的趋化效应及促CCR3-EGFP聚集的能力,且随着硫酸软骨素分子质量的增加,抑制作用显著增强。结论 GAGs的存在可以显著调控CCL11的聚集状态,进而影响其与受体CCR3的相互作用,本研究为进一步阐明CCL11-GAGs-CCR3相互作用关系提供了一定的实验基础。

Study on The Interaction Between Chemokine CCL11-glycosaminoglycan-chemokine Receptor CCR3

Objective To illustrate the interaction process and mechanism of chemokine CCL11 with receptor CCR3 and glycosaminoglycans (GAGs) and shade light on the regulation mechanism of CCL11-GAGs-CCR3.Methods A stably transfected CCR3-EGFP CHO cell line with single-molecule expression level was constructed using genetic engineering. The interaction between GAGs and CCL11 under different in vitro solution was studied by total internal reflection fluorescence (TIRF) and isothermal titration calorimetry (ITC) technique. The regulation of GAGs-CCL11 on chemotactic behavior of CCR3-EGFP stable cells and the effect of CCR3-EGFP aggregation on cell membrane were investigated by chemotactic assay and living cell monomolecular imaging assay.Results With the increase of chondroitin sulfate chain length, the binding heat release of chondroitin and CCL11 increased, indicating that the interaction force was enhanced and the promotion effect of CCL11 aggregation was enhanced. Single molecule fluorescence imaging technology combined with chemotactic test showed that different types and proportions of GAGs could affect the interaction between CCL11 and CCR3. The addition of GAGs inhibited the chemotactic effect of CCL11 on CCR3-EGFP stable cells and promoted the aggregation ability of CCR3-EGFP. With the increase of molecular mass of chondroitin sulfate, the inhibitory effect was significantly enhanced.Conclusion GAGs can affect the aggregation state of CCL11 and its interaction with receptor CCR3. Our study provides a theoretical basis for further elucidating the interaction between CCL11-GAGs-CCR3.