规模化猪场保育舍内NH3和 H2S恶臭气体刺激损伤猪呼吸道黏膜屏障的分子机制研究

Respiratory mucosa plays important physical and immunological barrier roles in maitaining the airway health.With the intensive pig production, the respiratory disease of nursery piglets induced by odorous and hamful gases in the pig housing is becomg a severe problem for pig production. As NH3 and H2S has been proved to be the two main irritant odorous gases to respiratory mucosa, in this project, we try to analyze the relationship between NH3 and H2S and respiratory disease. Specifically, the objectives of this of this project are: (1) Survey the NH3 and H2S concentrations, exposure time and related environmental and production parameters and information collection of respiratory disease morbidity in multiple nursery housing in central China for different pig breeds to find links between air quality and environmental/production parameters with respiratory disease. (2) Conduct controlled experiments with piglets exposed to certain concentrations of NH3 and H2S to investigate how NH3 and H2S affect the epithelial structure, mucin secretion, as well as the immune system of the respiratory mucosa on the basis of histology and cytology studies by using tissue section, immunohistochemistry, western blotting, RNAseq and real-time PCR technologies. The identified differentially expressed genes are beneficial to understand the molecular mechanism of injuring respiratory mucosa barriers. (3) Identify resistant or susceptible genes to respiratory disease (by using genotyping by genome reducing sequencing (GGRS) method) so that they may be used as molecular markers for improved pig breeding and disease-resistant selection. We assembed a multidisciplinary team of animal genetist, veterinary swine specialists, and air quality specilists to address these challenges on fundamental and practical levels. Proposed project will result in new fundamental knowledge about molecular mechanisms responsible for injuring respiratory mucosa. Molecular markers will be used for improved breeding and selection of disease-resistant lines.It will also provide information about air quality and envioronmental data associated with intensive pig production. Results will help the swine industry, improve food safety and security, and provide the veterinary and public health officials with improved tools and knowledge how to control the spread of respiratory disease.

在规模化猪场中，保育舍内恶臭气体诱发的仔猪呼吸道疾病不容忽视。本研究拟从保育舍内对呼吸道黏膜具有强烈刺激性的两种主要恶臭气体NH3和 H2S入手，在对我国华中地区多个规模化猪场保育舍内恶臭气体排放和仔猪呼吸道疾病发病情况进行调查的基础上，以一定浓度NH3或 H2S刺激鼻黏膜免疫具有差异的长白和鄂西黑猪构建呼吸道疾病模型；采集刺激后猪呼吸道黏膜组织，利用组织切片、免疫组化、RNAseq等技术从分子水平和细胞水平研究气体刺激对猪呼吸道黏膜组织形态、粘液分泌、免疫细胞和基因表达的影响，并对部分品种间差异表达基因进行深入的功能分析，试图解析猪呼吸道黏膜屏障的遗传基础；同时，结合基因组简化测序技术筛选可用于猪早期选种的抗恶臭分子标记。因此，本研究结果为猪场环境控制提供基础数据，为揭示舍内恶臭气体与猪呼吸道健康的内在联系提供理论依据，同时，在猪病防制及生物和食品安全方面又具有十分重要的应用前景。

本研究对我国华北、华中和华南三个规模化猪场秋、冬季节保育舍内NH3 和 H2S浓度及其它环境因子进行了检测与分析，初步获得了规模化猪场保育舍内NH3 和 H2S的浓度范围及变化规律。随后，利用80ppm氨气刺激保育仔猪0天（NC），4天（D4），8天（D8）和12天（D12），采集血液，鼻黏膜、肺脏、肝脏等组织，利用组织切片、免疫组化、ELISA、RNA-seq和定量PCR等技术，从组织学和分子水平解析了急性NH3刺激对仔猪呼吸道结构和基因表达的影响。主要研究结果如下：（1）研发了一套基于无线传感网络的猪舍环境监测系统，可对猪舍内氨气、硫化氢和二氧化碳浓度以及温湿度、照度进行实时监测；（2）制备了一个环境多变量控制养殖箱，能自动控制箱体内的氨气浓度和温度，可为氨气刺激相关试验提供设备保障；（3）初步获得了多个规模化猪场保育舍内NH3 和 H2S的浓度范围（0-40ppm和0-1ppm），发现氨气浓度与舍外温度呈显著的负相关；（4）发现80ppm氨气急性刺激造成保育仔猪鼻黏膜增厚，肺部炎性细胞渗出，肺上皮细胞-间质细胞的转化和肺轻度纤维化以及呼吸道黏液分泌增多，且鼻黏膜的损伤随着氨气刺激时间的延长逐渐加重，而肺组织的损伤则在 D4天表现最为严重，随后逐渐减轻并接近正常水平，揭示了上、下呼吸道对氨气刺激的响应表现不同；（5）发现80ppm氨气刺激引起保育仔猪鼻黏膜纤毛结构相关基因大量下调表达，肺组织炎症和先天性免疫相关基因大量上调表达，肝脏组织大量转录因子上调表达，暗示氨气刺激可能引起了仔猪呼吸道组织的炎症反应以及肝脏组织氧化应激和代谢的紊乱；（6）初步确定CXCL8，AGR2和TFF2可能作为氨气刺激后仔猪肺部急性炎症的标志基因，同时鉴定了一批差异表达的lincRNA，并通过WGCNA方法构建了差异表达mRNA和lincRNA的显著性模块，找到了肺脏和鼻黏膜D4、D8、D12以及肝脏D12特异高表达的基因模块，这些模块中的基因（包括lincRNA）可能在机体应对氨气刺激过程中发挥重要调控作用；（7）依托该项目，已发表通讯作者期刊论文3篇，申请、授权专利3项，培养硕士研究生6名。