DEC-205受体介导的树突状细胞靶向杜仲多糖纳米粒的佐剂活性及分子机理研究

Eucommia ulmoides Oliv polysaccharides (EUPS) can serve as an adjuvant to activate the immune system and enhance the effects of vaccination on specific and non-specific immunity in our previous study. but its disadvantage is that the effect time is short. Recently, we confirmed that DEC-205 receptor mediated dendritic cells targeting EUPS nanoparticles enhances the adjuvant activity of (EUPS). Thus, the immune-enhancing effect of polysaccharides act as a highly efficient new-type immunopotentiator should be enhanced. In this study, The DEC-205 receptor mediated dendritic cells targeting EUPS nanoparticles was prepared by orthogonal design. Firstly, the efficiency of uptake of nanoparticles, and the location of nanoparticles in the DC was investigated in vitro. In addition, the effect of the nanoparticles on maturation, cytokine secretion and Transcriptomics of DC was evaluated. Secondly, The immune-enhancing effects of nanoparticles in vivo were investigated through a series of experiments. The relative down-stream signaling pathway and acting target by which nanoparticles regulated the immune system was measured by molecular techniques. The molecular mechanism on the level of mRNA and protein by which nanoparticles activated the immune system was determined by cellular and molecular techniques. We explored the use of DEC-205 receptor mediated dendritic cells targeting EUPS nanoparticles modification in improving the immune-enhancing activity of polysaccharides and determined which nanoparticles exhibited the excellent results. Our results provide new theoretical evidence for the development of a novel immunopotentiator which can targeting DC cell. This new idea will be of great significance in developing novel veterinary drugs, promoting the prevention and control of major animal infectious diseases, and in protecting animal husbandry.

本实验室前期的研究发现川杜仲多糖（EUPS）作为佐剂可有效提高疫苗的免疫效果，但其缺点是作用时间较短。近期预实验发现通过树突状细胞（DC）特异性表达的DEC-205受体修饰EUPS并与PLGA制成可靶向DC的纳米粒，能显著提高EUPS的佐剂活性并延长其作用时间，在此基础上本项目拟通过正交法制备DEC-205受体修饰的EUPS包衣的PLGA纳米粒，首先通过体外试验研究DC对纳米粒的摄取效率及在DC内的定位，并研究其对DC成熟、分泌及转录组学的影响。其次，通过体内试验研究该纳米佐剂在动物体内的DC靶向性及对疫苗免疫反应的调控作用，并进一步探讨其下游信号转导通路及作用靶点，从细胞层面、转录和翻译水平充分揭示该纳米粒对动物免疫应答调控的分子机制。本研究不仅可为研制新型靶向树突状细胞的多糖类长效兽用免疫佐剂奠定基础，而且对开辟新药源、提高重大动物疫病的防控效果具有巨大的实用价值和重要的学术意义。

DEC-205受体是存在于抗原提呈细胞表面的一种参与抗原识别及吞噬的受体。基于此受体的功能，我们设计了靶向DEC-205受体的杜仲多糖纳米粒并评价其佐剂活性。本项目在前期研究的基础上，优选了该纳米粒的制备工艺参数，研究了其对OVA及口蹄疫疫苗的佐剂活性及分子机制。本项目完全按照计划进行，主要研究结果如下：（1）通过正交试验优选出了合成DEC-205受体靶向川杜仲多糖纳米粒的关键工艺参数。发现制备的纳米粒能够体外缓释，粒径均匀，分散特征良好，电位稳定，微观形貌为球形，表面光滑，外观完整。为靶向纳米粒的制备标准提供了参考数据。（2）该纳米粒显著提高树突细胞和巨噬细胞对抗原的吞噬活性、促进了细胞因子分泌。可能通过TNF信号通路、NF-κB信号通路、细胞因子-细胞因子受体相互作用和Toll样受体信号通路对APC发挥调控作用。（3）DEC-205靶向纳米粒协同OVA免疫小鼠，显著提高抗体水平、增强脾细胞增殖、诱导NK、CTL细胞毒性、增强DC成熟。该纳米粒能靶向淋巴结、脾脏等免疫器官，控制佐剂和抗原在注射点的缓慢释放，并具有优异的组织相容性和低毒性。（4）DEC-205受体靶向杜仲多糖纳米粒协同口蹄疫疫苗免疫小鼠。显著上调了FMDV特异性IgG水平、CTL和NK细胞的杀伤活性、脾细胞的增殖、T细胞亚群、巨噬细胞的吞噬能力、Th1和Th2型细胞因子的分泌，促进了DC成熟。（5）靶向DEC205受体的蕨麻多糖纳米粒的免疫活性研究表明，在体外该纳米粒能促进巨噬细胞吞噬活性及M1型极化基因敲除及受体饱和试验证明DEC205受体与抗原摄取过程相关。在体内显著提高受免动物细胞免疫、体液免疫与细胞因子分泌。DEC205-PAPP能够实现缓释与淋巴器官的靶向。.本项目研究表明，DEC205靶向纳米粒的佐剂活性显著优于非靶向纳米粒，DEC205受体靶向技术可为研发新型兽用靶向纳米佐剂提供有价值的理论依据，具有潜在的实用价值和广阔的应用前景。本项目作为第一资助已发表论文15篇，其中SCI论文12篇，中文论文3篇，还有1篇SCI论文在评审；作为第二资助已发表SCI论文1篇，获得国家授权实用新型专利4项，正在申报发明专利1项；线上及线下参加国内学术会议6次；培养研究生8名，本科生20名。