基于扶正祛邪法研究口服马齿苋多糖的FMD疫苗佐剂作用及其吸收机制

The outbreak of foot-and-mouth disease (FMD) has led to serious damage to animal husbandry. Vaccination failed to provide enough immune protection. Therefore, it is great significance to study FMD vaccine adjuvants for enhancing the immunogenicity. Heat-clearing and detoxifying method is an important means of Chinese Veterinary Medicine for FMD. The preliminary study has found that oral administration of polysaccharide (POL-P3b) extracted from Portulaca oleracea L., a kind of heat-clearing and detoxifying Chinese medicine, could improve the body’s immune function and reduce the toxin in vivo. According to the Fuzheng Quxie characteristics of POL-P3b, and combined with previous research, this project intends to elucidate the activities of POL-P3b as FMD vaccine adjuvants and the mechanism of enhanced immune response. This research is mainly to dendritic cells (DC), which is the important starter in the immune response. The techniques and methods, involving in flow cytometry, ELISA, immunohistochemistry, qRT-PCR, western blot and chromatin immunoprecipitation assay (ChIP), will be used to confirm that POL-P3b had activities of regulating the immune function. The digestion and absorption mechanism in vivo for macromolecular polysaccharide adjuvants still exists many the bottleneck problem of existing theory fails to explain. This study further to reveal the absorption mechanism of POL-P3b on small intestinal cells in vivo and in vitro by high performance liquid chromatography (HPLC) , fluorescent labeled polysaccharide and fluorescence resonance energy transfer（FRET）techniques. These data will provide theoretical basis for the absorption study of oral polysaccharides adjuvants in the body.

口蹄疫（FMD）的爆发给畜牧业带来严重危害。接种疫苗未能提供足够的免疫保护力。因此，研究FMD疫苗佐剂以提高其免疫原性具有重要意义。清热解毒法是中兽医治疗FMD的重要手段。前期研究发现，口服清热解毒中药马齿苋的活性成分多糖（POL-P3b）能提高机体免疫力、降低体内毒素。本项目拟对POL-P3b扶正祛邪特点，结合前期研究，针对免疫应答的主要启动者树突状细胞（DC），综合应用流式细胞术、ELISA、免疫组化、qRT-PCR、Western blot 和ChIP等方法，阐释POL-P3b的FMD疫苗佐剂作用及增强FMD疫苗免疫应答的机制。由于口服大分子多糖佐剂体内消化吸收机制仍然存在许多现有理论未能解释的瓶颈问题，本研究进一步通过高效液相色谱法（HPLC）、多糖荧光标记和荧光共振能量转移（FRET）等技术，体内外实验揭示小肠细胞摄取POL-P3b的机制，为研究口服多糖佐剂体内吸收提供理论依据。

口蹄疫（FMD）的爆发给畜牧业带来严重危害。接种疫苗未能提供足够的免疫保护力。研究FMD疫苗佐剂以提高其免疫原性具有重要意义。本项目在中兽医学基本理论指导下，利用本实验室制备的马齿苋多糖（POL-P3b），通过ELISA检测血清特异性IgG抗体及其亚类和CCK8法检测淋巴细胞增殖，明确了口服POL-P3b对FMD疫苗的免疫增强作用；通过ELISA检测肠道内sIgA，提示口服POL-P3b能激活肠道黏膜免疫系统，进而提高机体全身免疫应答。评价受免后小鼠免疫器官指数，结果提示，POL-P3b对免疫小鼠的胸腺和脾脏组织发育有一定的促进作用。病理形态学结果显示POL-P3b无毒副作用。针对免疫系统中发挥关键作用的免疫应答的主要启动者DC，基于流式细胞术、ELISA和qRT-PCR方法明确口服POL-P3b对FMD疫苗受免小鼠DC分化成熟及功能的影响。成熟DC通过MHC类分子、共刺激分子给T细胞提供活化所需的双信号，在细胞因子的共同作用下，激活T细胞，介导免疫应答，且POL-P3b影响DC的成熟和功能与线粒体功能和膜完整性密切相关。进一步通过免疫组化法检测TLR2、TLR4及下游分子MyD88和NF-κB的表达，揭示POL-P3b通过刺激TLR-4信号通路诱导DC成熟。通过Western Blot和ChIP技术深入探讨POL-P3b对DC功能细胞因子IL-12的表观遗传调控影响，明确DC的IL-12启动子区域组蛋白H3乙酰化水平与POL-P3b诱导的免疫活性密切相关。通过体外小肠模拟Caco-2模型和体内动物实验证实POL-P3b进入上皮细胞呈时间和浓度依赖型。POL-P3b可借助clathrin蛋白进入肠道细胞内，进而随血液到达全身各处，与其受体结合发挥各种生物功能，明确POL-P3b的口服吸收机制。本项目研究成果未来可应用于研发中药多糖佐剂领域。