中药多糖提高鸡ND疫苗呼吸道黏膜免疫的作用及机理研究

Respiratory mucous membrane immune system is particularly important for the prevention and control of animal epidemic disease, and at present the mucosal immune system research mainly focused on the intestinal mucosal immunization, little research on respiratory mucous membrane immune function was not enough in-depth, and its mechanism is not clear. Previous studies have found that mulberry leaf polysaccharide (MLP) can not only increase intestinal mucosal immune function, but also have significant advantages on improve the respiratory mucosal immune function. Based on the preliminary studies, chickens will be orally administrated with MLP at the same time of immunizing with ND vaccine by nose and eyes, the changes of mucous membrane of respiratory tract secretions will be assayed by ELISA methods, and the respiratory tract mucosa inherent lymphocytes will be observed by immunohistochemistry and flow cytometry methods. The effect of MLP on the respiratory mucous membrane of chickens which immunized with ND vaccine will be evaluated comprehensively according to the changes of humoral immunity and cellular immunity. The changes of genes and protein expression of TLRs in different tissue of respiratory and the number of DC and ILC2 on the respiratory tract mucosa inherent lymphocytes and related cytokines will be measured by Real-time PCR, Western Blot methods and flow cytometry, the results will reveal that the MLP is how to enhance the respiratory mucous membrane immune function, and illuminate the mechanism from the angle of the signal transduction. This study can not only provide research idea for respiratory mucous membrane immune effect and mechanism, but also would have a very good practical significance for prevention and control the animal disease especially virus which spread by respiratory.

呼吸道黏膜免疫系统对于动物疫病防控特别重要，而目前黏膜免疫研究主要侧重于肠道，对呼吸道黏膜免疫功能研究甚少且不够深入，其作用机理亦不明确。前期研究发现中药多糖不仅增强肠道黏膜免疫功能，在增强呼吸道黏膜免疫功能上也具显著优势。本项目以前期研究为基础，选择桑叶多糖、杜仲多糖、泡桐花多糖为试验药物，在鸡免疫ND疫苗同时口服上述中药多糖，以呼吸道黏膜分泌液和固有淋巴细胞为研究对象，采用ELISA、免疫组化和流式细胞术，从体液和细胞免疫水平上比较不同中药多糖对ND疫苗呼吸道黏膜免疫的调节作用，筛选出最佳的中药多糖。采用流式细胞术、RT-PCR、Western Blot等分子生物学技术，从基因和蛋白表达水平探讨在呼吸道不同组织部位中药多糖对TLR、DC和ILC2作用，并从信号转导角度阐明调节的分子机制。本研究不仅为呼吸道黏膜免疫作用及机制提供研究思路，而且对于经呼吸道传播的病毒性疾病防控具有重要意义。

呼吸道黏膜免疫系统在免疫防御中发挥了重要的作用。本研究首先通过不同中药提取物的免疫调节作用比较，筛选出桑叶粗多糖作为呼吸道黏膜免疫研究的中药成分。后对桑叶多糖进行纯化、鉴定确定了MLP-2具有更强的免疫调节活性。然后开展了纯化的桑叶多糖（简称桑叶多糖）对免疫ND疫苗雏鸡呼吸道黏膜免疫的调控作用及机理研究。结果显示，桑叶多糖可显著提高血清NDV特异性抗体效价及IgG和IgA亚类水平，显著促进气管和支气管肺泡灌洗液中sIgA和IL-2、IFN-γ相关免疫细胞因子的分泌和气管、支气管、肺组织中IgA mRNA表达水平。表明桑叶多糖可诱导免疫NDV雏鸡产生较高的呼吸道黏膜抗体应答，尤其作用于气管。桑叶多糖可显著促进气管杯状细胞的产生以及气管黏蛋白MUC5AC、MUC5B和紧密连接蛋白ZO-1、occludin和claudin1的mRNA表达水平，提示桑叶多糖参与免疫ND疫苗雏鸡气管黏膜屏障功能的调节，增强呼吸道黏膜屏障功能。在细胞免疫应答方面，桑叶多糖可显著促进NDV免疫雏鸡T淋巴细胞亚群的扩增，增强骨髓DCs吞噬功能、促进DC表面CD40和CD86分子的表达，促进DCs分泌IL-1β、IFN-γ、IL-12p70、TNF-α与RANTES等细胞因子，抑制炎症状态下IL-10的分泌；通过建立鸡肺巨噬细胞的分离、培养和鉴定方法，开展了桑叶多糖对鸡肺巨噬细胞的调节作用，结果表明桑叶多糖体外能激活巨噬细胞，增强其吞噬能力，促进巨噬细胞分泌IL-2、TNF-α和IFN-γ等免疫细胞因子，显著增强鸡肺巨噬细胞分泌NO的作用。在呼吸道黏膜免疫调控机制研究方面，发现TLR7可能参与桑叶多糖增强免疫NDV雏鸡的呼吸道黏膜免疫。桑叶多糖对呼吸道巨噬细胞体外调控机制表明，桑叶多糖通过激活巨噬细胞TLR4受体MyD88依赖性和TRIF依赖性两条信号途径发挥免疫调节作用。本研究揭示了中药多糖对鸡呼吸道黏膜免疫的调节作用，阐明了其可能的调控机制，为今后进一步开展鸡呼吸道黏膜免疫的研究提供了研究基础。同时也为桑叶多糖开发为免疫调节剂提供了理论依据。