猪繁殖与呼吸综合征病毒感染的猪肺中eNOS及其相关因子表达量变化的研究

发布时间：2018-03-19 19:41

本文选题：猪繁殖与呼吸综合征　切入点：肺损伤　出处：《华中农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：猪繁殖与呼吸综合征(PRRS)是一种传染性极强的猪病,临床主要表现为流产、早产等繁殖障碍和不同程度的呼吸症状,如呼吸困难、肺炎等。病死猪剖检通常可见肺部出现弥漫性间质性肺炎并伴有大量出血点,因此肺部病变导致的肺损伤是造成猪死亡的主要因素之PRRSV感染猪的过程中,肺损伤的形成可能与血管内皮细胞形态和功能的改变关系密切。一氧化氮(NO)是可以改变内皮细胞的形态和功能的因素之一,而内皮型一氧化氮合酶(eNOS)控制着血管内皮细胞中NO的合成。因此,eNOS及其相关因子的含量变化可能会与PRRSV感染猪造成肺损伤有所关联。本实验旨在通过对PRRS中的eNOS及其相关因子变化的研究,阐明猪PRRS中肺脏炎症反应及肺损伤的相关分子机理,为PRRS的预防与治疗提供思路。本实验所用样品均为大白猪猪肺,分为三组：正常对照猪肺样本、临床PRRS猪肺样与人工攻毒PRRS猪肺样。通过荧光定量PCR对比了eNOS与iNOS在PRRS病猪中的变化；Western blot对比了eNOS与微囊蛋白-1的含量变化；分别通过硝酸还原酶法与化学荧光法检测了肺组织内NO和活性氧簇(ROS)含量变化。通过苏木精-伊红染色和免疫组化对比血管内皮细胞形态变化和eNOS蛋白含量变化。结果显示,与正常对照组相比,临床组与攻毒组中eNOS的mRNA相对表达量显著升高(P0.05); iNOS的mRNA变化不显著(P0.05)；eNOS蛋白表达量显著升高,Cav-1蛋白表达量临床组中升高；NO含量显著降低(P0.05); ROS含量变化不显著(P0.05)；病理组织学结果表明,与正常猪相比,PRRS病猪肺中的大血管结构破损,毛细血管管径变小并且数目减少；免疫组化中eNOS蛋白显著升高(P0.05)。研究结论：1.临床感染的PRRS病猪与攻毒的PRRS病猪在实验结果上呈现出一定的符合性；2. PRRSV的感染可以导致病猪肺内的NO含量下降,而eNOS的mRNA和蛋白均上升,表明PRRSV感染可能导致eNOS的活性降低,临床PRRS中与eNOS结合的Cav-1蛋白增加,可能是eNOS的活性降低的因素之一。3.iNOS和ROS在PRRS病猪肺组织中的变化个显著,说明在PRRS中,iNOS对NO的调节作用并不明显,NO减少的同时并未造成ROS堆积。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRSs) is a highly infectious disease of pigs. Its clinical manifestations are abortion, premature birth and other reproductive disorders, as well as various respiratory symptoms, such as dyspnea. Lung injury caused by lung disease is the main factor leading to pig death. PRRSV infection is the main cause of pig death. The formation of lung injury may be closely related to the changes in the morphology and function of vascular endothelial cells. Nitric oxide (no) is one of the factors that can change the morphology and function of endothelial cells. Endothelial nitric oxide synthase (Enos) controls the synthesis of no in vascular endothelial cells. Therefore, the changes of Enos and its related factors may be associated with lung injury in pigs infected with PRRSV. This study was designed to investigate the effect of eNOS in PRRS on lung injury in pigs. And its related factors, To elucidate the molecular mechanism of pulmonary inflammation and lung injury in pig PRRS, and to provide ideas for the prevention and treatment of PRRS. The samples used in this study were all large white pig lungs, which were divided into three groups: normal control pig lung samples. The changes of eNOS and iNOS in PRRS infected pigs were compared by fluorescence quantitative PCR. The contents of eNOS and microcapsule protein 1 were compared by Western blot. The contents of no and Ros in lung tissue were detected by nitrate reductase method and chemical fluorescence method respectively. The morphological changes and eNOS protein contents of vascular endothelial cells were compared by hematoxylin eosin staining and immunohistochemistry. Compared with the normal control group, The relative expression of eNOS in clinical group and drug attack group was significantly higher than that in control group (P 0.05), but the mRNA expression of iNOS was not significant. The expression of Cav-1 protein was significantly increased in clinical group. The content of no was significantly decreased in clinical group, the content of ROS was not changed significantly (P 0.05); the pathological changes were not significant. Histologically, Compared with the normal pigs, the pulmonary macrovascular structure of PRRS pigs was damaged, and the diameter of capillaries became smaller and the number of capillaries decreased. The expression of eNOS protein increased significantly in immunohistochemistry. Conclusion: 1. The clinical infected PRRS pigs and the infected PRRS infected pigs showed a certain conformance with the experimental results. The infection of PRRSV could lead to the decrease of no content in the lung of the infected pigs. However, both mRNA and protein of eNOS increased, indicating that PRRSV infection may lead to the decrease of eNOS activity, and the increase of Cav-1 protein binding to eNOS in clinical PRRS may be one of the factors that contribute to the decrease of eNOS activity. 3. The changes of eNOS and ROS in the lung tissue of PRRS pigs are significant. The results showed that the effect of iNOS on no in PRRS was not obvious and no was decreased and ROS accumulation was not caused.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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