氨气与空气湿度互作效应对仔猪肺泡上皮功能的影响及机制研究

Gas exchange happens in the alveolar, and the normal function of alveolar epithelium is important for lung function. Ammonia and the relative humidity(RH) in the building of nersery piglets were the two important envionmental factors that affect the lung function of animals. In this study, we will set up 3×3 factorial experimental design with three RH levels combined three ammonia concentrations. Six Piglets for each group are put in an environment multi-variable control aquaculture test box for environmental exposure under the different experimental conditions,respectively. The blood samples are collected on day 0, 3 and 30. The injury score of the airway tissues are recorded after the piglets are executed on day 30 and the lung tissues are collected for tissue morphology observation, and RNA/proteins isolation.The function of alveolar epithelium (permeability, fluid transport, pulmonary surfactant secretion) are deteceted by ELISA, immunofluorescence, western blot, Flow cell separation, electronic microscope. And a general linear mixed model will be used to evaluate the effect of ammonia, RH and their interaction on the function of alveolar epithelium in piglets. In the same time, the genome-wide gene expression and transcriptional regulatory element are detected by RNA-seq and ATAC-seq to explore the molecular mechanisam of ammonia and RH effects on the function of alveolar epithelium. The results will benefit the further understanding of the mechanism of envionment effects on animal health and may also offer new theoretical foundation for envionment control.

正常肺泡上皮功能是肺进行有效气体交换的重要保障。猪舍内的氨气浓度和空气湿度是影响仔猪肺部健康的两个重要环境因子，且氨气浓度和理化性质受到空气湿度影响。为探讨这两个因子及其互作对仔猪肺泡上皮功能的影响，本研究拟采用3×3析因试验设计，设置不同水平氨气浓度和空气湿度组合，对仔猪进行环境暴露刺激。通过ELISA、western、流式细胞分选和透视电镜观察等技术，检测不同处理组仔猪肺泡上皮功能（上皮通透性、液体转运功能、分泌功能）变化，利用广义线性混合模型分析氨气和空气湿度及其互作效应对仔猪肺泡上皮功能的具体影响。同时结合RNA-seq和转座酶的染色质可进入性的高通量测序（ATAC-seq）技术，分析处理前后肺组织全基因组水平基因表达变化，鉴定相关转录调控元件，在分子水平解析氨气和空气湿度影响仔猪肺泡上皮功能相关机制。研究结果为进一步解析猪舍环境对仔猪健康的影响以及指导猪舍环境控制提供理论依据。

猪舍内的氨气浓度和空气湿度是影响仔猪肺部健康的两个重要环境因子，本研究设置不同水平氨气浓度和空气湿度组合，对仔猪进行30天环境暴露刺激试验。通过ELISA、western、组织切片、免疫荧光、透视电镜观察结合转录组，蛋白质组，代谢组和微生物组测序等技术，检测不同处理组仔猪肺泡上皮结构与功能变化，从个体、组织、细胞和分子等多个水平解析氨气浓度和空气湿度对仔猪肺泡上皮的影响。取得主要研究结果如下：（1）肺组织HE染色结果和电镜结果显示，85%RH+20ppm氨气浓度下，肺泡结构相对完整，局部出现肺泡隔增厚现象，AECⅡ染色质均匀分布；85%RH+50ppm氨气浓度下，肺部出血点多，肺泡隔增厚，肺泡数目急剧减少，AECⅡ发生凋亡。（2）转录组结果显示，85%RH+20ppm氨气浓度下，T细胞介导的促炎反应和氧化应激反应被激活；85%RH+50ppm氨气浓度下氧化还原反应、脂质运输、离子转运等过程相关基因表达发生显著改变，DNA修复和凋亡过程相关基因表达下调，高氨暴露诱导肺泡上皮损伤。（3）蛋白质组结果显示，85%RH+50ppm氨气浓度下，肺组织中脂肪酸β氧化和促纤维化通路被激活，肺泡表面活性蛋白分泌下降，参与能量代谢，蛋白质合成和信号转导的蛋白质赖氨酸乙酰化修饰状态发生显著改变，进一步通过试验验证了黏着斑蛋白ILK K191乙酰化通过下调p473-AKT水平抑制A549细胞的增殖。（4）代谢组和微生物组结果显示，氨气刺激导致肺脏组织的有氧氧化减弱，脂质分解加剧，与蛋白质组结果吻合。氨气刺激后菌群结构发生显著变化，与肺部疾病相关的菌群数目增多，假单胞菌属丰度在高氨组显著增多，肠球菌属的丰度在氨气刺激后则显著减少。（5）低氨浓度下，不同空气湿度对肺脏组织结构影响不明显，但是基因表达存在差异。对相对湿度分别为65%和85%，氨气浓度为0-5ppm的两个组进行转录组比较分析发现，差异表达基因主要与细胞内稳态、物质转运和离子通道相关。（6）依托该项目，已发表通讯作者SCI论文5篇，培养毕业硕士研究生3名，在读研究生4名；参加国内国际学术会议并提交摘要4份，做分组报告1次。

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