双重靶向APCs的载壳聚糖PLGA纳米微球增强黏膜免疫的分子机制

Induction of specific mucosal immunity is essential for the clearance of pathogens in mucosal infections. The development of a new vaccine delivery system that targets antigens to antigen-presenting cells(APCs) in the mucosae is of great value for enhancing immune responses. Previous studies have shown that chitosan-coated PLGA nanoparticles loaded with plasmid DNA from foot-and-mouth disease(FMD) virus have a strong effect on mucosal immunity, and also antiviral effects. The improvement of targeted delivery of nanoparticles and the analysis of the immune response mechanism involved need further study. Based on APCs surface-specific receptors, mannose-modified chitosan-coated PLGA nanoparticles, loaded with DNA coding for FMD multi-epitope genes as model antigens, contained cytokine Flt3 ligands, will be designed to achieve dual APCs targeting. Through the analysis of physicochemical properties and the characterization of biological responses, specific nanoparticle targeting in vivo and in vitro, nanoparticle uptake by antigen-presenting cells, and nanoparticle functional mechanisms will be investigated. The effect of nanoparticles on the expression of receptors of DCs and macrophages, and on the activation of signaling pathways will be investigated. The effect of nanoparticles on mucosal immunization in mice will also be investigated. Our results will reveal the mechanisms involved inactivating antigen presentation and immune responses by targeted DNA-absorbed nanoparticles and also provide a foundation forresearch on the application of targeted nanoparticles as delivery system for mucosal antigen-presenting cells.

诱导特异黏膜免疫对黏膜感染病原体的清除非常关键。开展新型可将抗原靶向递送至黏膜抗原提呈细胞(APCs)的疫苗递送系统的研究，对增强免疫应答效果有重要应用价值。前期研究发现吸附口蹄疫DNA疫苗载壳聚糖PLGA纳米微球有较强的黏膜免疫效果和抗病毒保护，但是，如何增强纳米微球的靶向递呈以及解析其免疫反应的机制，尚待进一步研究。基于DCs表面特异性受体以及细胞生长因子受体Flt3配体(Flt3 ligand,FL)，本研究以口蹄疫多表位基因的真核表达载体为模型抗原，设计包含FL的吸附DNA甘露糖修饰载壳聚糖PLGA纳米微球，达到主动性的双重靶向，考察APCs对纳米微球的摄取、体内外特异性靶向能力及作用机制；考察纳米微球对APCs的受体表达的影响及信号通路的激活；开展纳米微球对小鼠黏膜免疫效果评价，这将对揭示靶向性纳米微球激活机体抗原递呈和增强免疫应答的机理提供分子机制研究基础。

诱导特异黏膜免疫对黏膜感染病原体的清除非常关键。开展新型可将抗原靶向递送至黏膜抗原提呈细胞(APCs)的疫苗递送系统的研究，对增强免疫应答效果有重要应用价值。前期研究发现吸附口蹄疫DNA疫苗载壳聚糖PLGA纳米微球有较强的黏膜免疫效果和抗病毒保护，但是，如何增强纳米微球的靶向递呈以及解析其免疫反应的机制，尚待进一步研究。基于DCs表面特异性受体以及细胞生长因子受体Flt3配体(Flt3 ligand,FL)，本研究以口蹄疫多表位基因的真核表达载体为模型抗原，设计包含FL的吸附DNA甘露糖修饰载壳聚糖PLGA纳米微球，达到主动性的双重靶向。考察APCs对纳米微球的摄取、体外特异性靶向能力及作用机制；开展纳米微球对小鼠以及豚鼠黏膜免疫效果评价。结果如下：成功制备了低取代度6%~10%甘露糖修饰的壳聚糖PLGA纳米微球；确定了纳米微球与质粒DNA最佳质量结合比60:1并且纳米微球载质粒DNA可以在RAW264.7细胞中表达；体外验证了甘露糖修饰的纳米微球可被DCs细胞特异性摄取；小鼠免疫实验中，双重靶向疫苗组诱导小鼠产生更强的免疫反应；豚鼠攻毒保护实验表明双重靶向疫苗组对豚鼠经口蹄疫病毒攻击的保护效果最好。总之，该研究制备了稳定、高效的双重靶向APCs的载壳聚糖PLGA纳米微球，通过优化制备参数，建立一套通用的靶向性载壳聚糖PLGA纳米微球黏膜免疫递呈体系平台技术；明确了靶向性分子能够将抗原递送至APCs，增强纳米微球诱导的黏膜免疫应答，该研究对FMDV及经过黏膜系统传染和发病的烈性传染病的预防及控制具有重要的参考价值。