鼻腔黏膜是抵御病原微生物入侵呼吸系统的第一道防线，鼻腔黏膜屏障在抵御病原微生物入侵中发挥着重要作用。因此，增强鼻腔黏膜的屏障功能可有效预防或控制猪呼吸道疾病的发生。鼻腔黏膜从外向内由三层屏障构成：菌群屏障、黏膜上皮屏障（物理屏障）和免疫屏障，在猪的不同发育阶段具有较大差异。本研究从鼻腔黏膜菌群屏障、物理屏障和免疫屏障出发，揭示了不同日龄三元肉猪的鼻腔黏膜屏障特点，结果表明了哺乳期仔猪鼻腔屏障功能不完善，存在病原感染的潜在风险。因此，本研究使用了一株具有鼻腔定植能力的枯草芽孢杆菌NS15进行喷鼻保护，以有效增强哺乳期仔猪的鼻腔黏膜免疫力。结果表明喷鼻枯草芽孢杆菌NS15可以显著活化猪鼻腔黏膜下树突状细胞（Dendritic cells，DCs），增强DCs的免疫应答，包括增强DCs的迁移能力、抗原摄取、呈递能力。随后，本研究进一步具体阐明了枯草芽孢杆菌NS15活化猪鼻腔黏膜下DCs的相关机制，枯草芽孢杆菌NS15可以诱导呼吸道上皮细胞分泌趋化因子，通过激活微丝骨架信号通路使DCs的微丝骨架重排，从而募集鼻腔黏膜下的DCs形成跨上皮树突摄取抗原活化DCs。本研究具体内容分为以下三个部分：

1.不同日龄的猪鼻腔黏膜特点

鼻腔黏膜屏障由菌群屏障、上皮物理屏障和免疫屏障构成，本研究对猪不同日龄（包括新生期、哺乳期、断奶期、育肥期和育成期）鼻腔黏膜各屏障的结构特点进行了研究。结果表明猪鼻腔内的优势菌门为厚壁菌门（Firmicutes）、变形菌门（Proteobacteria）和拟杆菌门（Bacteroidetes），其中厚壁菌门的丰度从哺乳期开始随日龄的增加而显著下降，而变形菌门和拟杆菌门的丰度随日龄的增加而增加，在哺乳期鼻腔致病菌的丰度显著上升。通过菌群功能分析，我们发现随着日龄增加，菌群的存在有利于猪鼻腔黏膜的自身代谢和修复。我们还发现在猪的生长发育过程中，鼻腔前庭区上皮细胞层明显增厚，呼吸区和嗅区固有层腺体、杯状细胞数量明显增多。上皮细胞的紧密连接表达和增殖能力也具有明显的日龄依赖性，但在哺乳期有明显的下降，在断奶期得到恢复。在新生期和哺乳期，CD3+T细胞、IgA+B细胞和DCs在鼻腔黏膜分布数量较少，在断奶期显著增加。除此之外，大部分模式识别受体表达水平在断奶期也显著增加（P < 0.05）。综上，哺乳期的仔猪鼻腔屏障结构不完善，存在呼吸道感染的潜在风险。

2.猪源枯草芽孢杆菌活化猪鼻腔黏膜下树突状细胞的研究

为有效提升哺乳仔猪鼻腔黏膜免疫力，本研究使用一株分离自猪鼻腔的枯草芽孢杆菌（NS15）作为鼻腔免疫增强剂，结果显示喷鼻枯草芽孢杆菌NS15能显著增加鼻腔黏膜中DCs的数量（P < 0.05）。为了进一步探究猪源枯草芽孢杆菌NS15对鼻腔黏膜下DCs的作用，首先我们建立了猪呼吸道上皮细胞和DCs的气液共培养模型，随后使用流式细胞术和共聚焦显微镜对DCs的免疫功能进行检测。结果显示枯草芽孢杆菌NS15可以激活位于猪呼吸道上皮细胞下的DCs，增强DCs的迁移能力、抗原摄取和抗原递呈能力。进一步采用转录组学分析的方法检测了枯草芽孢杆菌NS15作用呼吸道上皮下的DCs基因转录的变化，结果显示猪源枯草芽孢杆菌NS15可以激活呼吸道上皮下DCs多个免疫相关信号通路，包括NF-kB、Toll样受体和C型凝集素信号通路等。同时我们还发现DCs中IL-6、IL-8、IL-12和IFN-γ等细胞因子的表达也显著性增加（P < 0.05）。综上，猪源枯草芽孢杆菌NS15可以活化猪呼吸道上皮细胞下DCs，但是其活化的具体机制需要进一步研究。

3.枯草芽胞杆菌通过调控猪呼吸道上皮细胞分泌趋化因子CCL20招募并活化树突状细胞的机制研究

为阐明枯草芽孢杆菌NS15活化猪呼吸道上皮下DCs的机制，对上皮细胞分泌的趋化因子进行了检测，结果显示枯草芽孢杆菌NS15能够显著增加上皮细胞趋化因子CCL20的表达和分泌（P < 0.05）。为了检测CCL20对DCs的趋化能力，使用共聚焦显微镜对DCs跨上皮树突的形成以及微丝骨架的变化进行观察，并使用Western blot对DCs微丝相关信号通路蛋白进行检测。结果表明趋化因子CCL20可以结合DCs上的趋化因子受体CCR6，激活微丝信号通路蛋白引起DCs的微丝骨架重排，从而有利于DCs跨越上皮细胞伸出树突来摄取抗原。

本研究首先指出了哺乳期仔猪存在鼻腔黏膜屏障功能不完善的问题，随后通过喷鼻具有定植能力的枯草芽孢杆菌以增强哺乳期猪鼻腔黏膜的免疫保护力，进一步探究了枯草芽孢杆菌NS15活化猪鼻腔黏膜DCs的机制，结果表明枯草芽孢杆菌NS15诱导呼吸道上皮细胞分泌趋化因子CCL20，导致DCs微丝骨架的重排，促进DCs跨上皮树突形成。综上，本研究揭示了枯草芽孢杆菌活化猪鼻腔黏膜中DCs的作用机制，为猪鼻腔黏膜免疫研究提供了重要的理论依据。

The nasal cavity mucosa is the first line of defense against the invasion of pathogenic microorganisms in the respiratory system, and the nasal mucosal barrier plays an important role in resisting the invasion of pathogenic microorganisms. Therefore, enhancing the barrier function of nasal mucosa can effectively prevent or control the occurrence of respiratory diseases in pigs. Nasal mucosa is composed of three barriers from the outside to the inside: bacterial barrier, mucosal epithelial barrier (physical barrier) and immune barrier, which have great differences in different developmental stages of pigs. This study from the nasal cavity flora barriers, physical barriers and immune barrier three levels, reveals the different growth stages of ternary nasal mucosal barrier characteristics of pigs, shows the suckling piglet nasal barrier function is not perfect, there is the potential risk of pathogen infection. In order to effectively enhance nasal mucosal immunity at this stage, this study used a strain of Bacillus subtilis NS15 with nasal colonization ability for nasal spray protection. Bacillus subtilis NS15 significantly activated submucosal Dendritic cells (DCs), including enhancing DCs migration ability, antigen uptake, presentation ability and immune response. Finally, this study specifically elucidated the relevant mechanism of submucosal DCs activation by Bacillus subtilis NS15. The results showed that Bacillus subtilis NS15 could induce pig respiratory epithelial cells to secrete chemokine CCL20, which combined with CCR6 receptor on DCs to activate DCs by activating the microfilament skeleton signaling pathway to rearrange the microfilament skeleton of DCs, so as to attract DCs in the nasal mucosa to form trans-epithelial dendritic uptake of antigen. The specific content of this study is divided into the following three parts:

1. Characteristics of nasal mucosa of pigs at different ages

The nasal mucosal barrier consists of biological barrier, mechanical epithelial barrier and immune barrier. This study was conducted to study the structural characteristics of nasal mucosal barriers in pigs at different growth and development stages, including neonatal stage, suckling stage, weaning stage, growing stage and finishing stage. The results showed that with the growth and development of pigs, the epithelial cell layer in the vestibular area of nasal cavity was significantly thickened, and the glands and goblet cells in the lamina of respiratory and olfactory areas were significantly increased. The expression of tight junction and proliferation of epithelial cells were significantly age-dependent. The number of CD3+T cells, IgA+B cells and dendritic cells involved in the nasal immune barrier increased significantly during weaning, and the mRNA expression levels of most pattern recognition receptor genes also increased significantly during weaning (P < 0.05). Firmicutes, Proteobacteria and Bacteroidetes were the dominant bacterial phyla in nasal cavity of pigs. The abundance of Firmicutes decreased significantly with age, while the abundance of Proteobacteria and Bacteroidetes increased with age, and the abundance of pathogenic bacteria in nasal cavity increased significantly during lactation. Through functional analysis, we found that with the increase of age, bacterial community has a positive effect on the metabolism and repair of nasal mucosa in pigs. It is important to note that the nasal barrier of suckling piglets is not perfect and there is a potential risk of respiratory infection.

2. Activation of submucosal dendritic cells from porcine nasal cavity by Bacillus subtilis

To effectively enhance the nasal mucosal immunity of suckling piglets, a strain of Bacillus subtilis NS15 isolated from the nasal cavity of pigs was used as nasal immune enhancer. The results showed that Bacillus subtilis NS15 significantly increased the number of DCs in nasal mucosa (P < 0.05). To further investigate the effects of porcine Bacillus subtilis NS15 on DCs, we established a gas-liquid co-culture model of respiratory epithelial cells and DCs, and tested the immune function of DCs by flow cytometry and confocal microscopy. The results showed that Bacillus subtilis NS15 could activate DCs located in epithelial cells and enhance the ability of migration, antigen uptake and antigen presentation of DCs. Furthermore, transcriptome analysis of the changes of gene expression level of porcine Bacillus subtilis NS15 in nasal submucosal DCs showed that porcine Bacillus subtilis NS15 could enhance the immune function of nasal submucosal DCs and activate multiple immune-related signaling pathways, such as NF-KB, Toll-like receptor signaling pathway. The expression of IL-6, IL-8, IL-12 and IFN-γ in DCs increased significantly (P < 0.05). In conclusion, porcine Bacillus subtilis NS15 can activate DCs in epithelial cells, but the specific mechanism of activation remains unclear.

3. Study on the mechanism of Bacillus subtilis recruitment and activation of dendritic cells by regulating pig respiratory epithelial cell secretion of chemokine CCL20

In order to elucidate the mechanism of activation of DCs in pig respiratory epithelial cells by Bacillus subtilis NS15, chemokine secreted by epithelial cells was detected, and the results showed that Bacillus subtilis NS15 could significantly increase the expression and secretion of epithelial chemokine CCL20 (P < 0.05).   In order to verify the chemotactic ability of CCL20 on DCs, confocal microscopy was used to photograph the trans-epithelial dendrites and microfilaments formed by DCs. The results showed that chemokine CCL20 could bind to chemokine receptor CCR6 on DCs to cause the rearrangement of the microfilaments. Subsequently, Western blot analysis of DCs filaments signaling pathway proteins showed that CCL20 could induce activation of the filaments signaling pathway, leading to the rearrangement of DCs filaments, which was beneficial for DCs to extend dendrites across epithelial cells to absorb antigens.

This study first lactation piglets are pointed out of the imperfection of the nasal mucous membrane barrier function. Subsequently, Bacillus subtilis NS15 was sprayed to enhance the immune protection of nasal mucosa of pigs during this period. The mechanism of DCs activation in porcine nasal mucosa by Bacillus subtilis NS15 was further explored, the secretion of chemokine CCL20 by respiratory epithelial cells to recruit and promote the formation of DCs trans-epithelial dendrites was further explored. In conclusion, this study revealed the mechanism of DCs activation by Bacillus subtilis in porcine nasal mucosa, and provided an important theoretical basis for porcine nasal mucosal immunity research. 

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