硒对金黄色葡萄球菌性乳腺炎发生的影响及调控机制研究

发布时间：2018-01-08 06:17

本文关键词：硒对金黄色葡萄球菌性乳腺炎发生的影响及调控机制研究　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：乳腺炎是严重危害奶牛健康的重要疾病。该病导致患牛泌乳量减少、奶牛淘汰率增加,同时引发相关食品安全问题。奶牛乳腺炎的发生与许多因素有关,其中病原感染是奶牛乳腺炎发生的主要原因。诸多报道显示多种微生物均可引发该病,包括病毒、寄生虫、支原体、真菌及细菌等等。金黄色葡萄球菌(金葡菌)是诱发奶牛乳腺炎的主要病原。然而,我们临床调查发现并不是金葡菌入侵的所有奶牛都发生乳腺炎。这些说明该病的发生与动物机体的自身免疫状态密切相关。众所周知,营养状态与哺乳动物的免疫能力密切相关。奶牛产后大量泌乳,众多营养物质损失,导致机体免疫力降低。此时乳腺组织对外界入侵病原防御能力降低,致病微生物感染引发乳腺炎。研究显示,缺硒促进泌乳早期乳腺炎的发生,增加日粮硒摄入可显著提高细胞吞噬活性并降低乳腺炎发生。大量的临床研究还发现,奶牛金葡菌性乳腺炎患病率与牛乳中硒含量呈现线性相关。日粮中适当补硒可减少乳腺病原菌感染,显著降低乳腺炎发病率,但其中的作用机制目前还不清楚。本研究以硒对乳腺局部免疫调控作用为切入点,探究其对金葡菌性乳腺炎发生影响并初步分析其作用机制。研究首先饲喂小鼠不同含硒浓度的鼠粮,然后建立金葡菌乳腺炎模型,同时分离乳腺上皮细胞进行相关研究。结果发现金葡菌感染导致乳腺组织结构被破坏,大量乳腺上皮细胞脱落死亡。缺硒饲喂小鼠炎性损伤最为严重,而富硒饲喂小鼠炎症变化相对轻微。对炎性因子进行分析发现,缺硒促进乳腺组织因金葡菌感染导致的IL-1β,TNF-α、IL-6,12、IFN-γ、NO等促炎因子分泌,抑制PPAR-γ,IL-10抗炎因子分泌。富硒饲喂可相对降低促炎因子分泌,增加抗炎因子分泌。我们分离乳腺上皮细胞,通过不同浓度含硒培养基孵育,给予金葡菌刺激后进行相应的研究,获得了与组织研究相一致的结果。我们进一步对金葡菌的主要识别受体TLR2及其下游炎症调控信号通路进行分析。结果发现,硒对NF-κB和MAPKs信号通路各分子基因表达水平没有影响,但添加硒可降低炎症发生后TLR2的过度表达,抑制其下游NF-κB及MAPK炎症信号通路的启动,降低炎性损伤。缺硒则会增加TLR2的过度表达,加重炎症反应。金葡菌性乳腺炎除表现炎症外,还会引发乳腺上皮细胞凋亡而破坏乳腺组织结构。为了全面解析硒的作用,我们通过在体及体外实验对细胞凋亡及损伤情况进行分析。结果显示硒对金葡菌诱导的乳腺组织细胞凋亡具有很好的调节作用。缺硒增加p53磷酸化,提高BAX/BCL-2值,促进Caspase-3、6、7蛋白裂解,加速金葡菌诱导的细胞凋亡程序启动。补硒可以抑制细胞凋亡程序启动,从而对乳腺组织发挥保护作用。众所周知,硒通过硒蛋白发挥其各种生物学功能。在诸多硒蛋白中已经证实硒蛋白S(Sel S)与炎症发生密切相关,为进一步深入研究硒对金葡菌乳腺炎发生的调控机制,我们以Sel S为靶点,通过RNA干扰技术进行深入研究。研究显示硒通过影响硒蛋白S的表达调控NF-κB和MAPK炎症信号通路各分子的蛋白磷酸化水平,调节炎症相关因子的分泌。硒可通过硒蛋白S可调控P53磷酸化水平,Bax,Bcl-2蛋白表达及Caspase-3、6、7凋亡执行因子蛋白裂解,从而对金葡菌乳腺炎的发生发挥调控作用。综上表明,硒对金葡菌性乳腺炎的发生发挥着重要调控作用。Sel S在这个调控过程中发挥着重要的作用。本研究为阐明金葡菌性乳腺炎的发病机制,寻求奶牛乳腺炎防治药物及有效措施提供新的靶点和思路。同时丰富硒的生物学作用,为营养免疫学在动物疾病方面的发展提供理论基础和帮助。
[Abstract]:Mastitis is a serious disease of cow health. The disease causes suffering from bovine milk yielddecreased cow culling rate increased, while related to food safety issues. Mastitis occurrence is related to many factors, including pathogen infection is a major cause of mastitis occurrence. Many reports show that many microorganisms can cause the disease, including the virus. Parasites, mycoplasma, bacteria and fungi. Staphylococcus aureus (S. aureus) is a major pathogen induced mastitis. However, our clinical investigation found that not Staphylococcus aureus invasion by cow mastitis. These instructions are closely related to the occurrence of the disease and animal immune state. As everyone knows, the nutritional status and the mammalian immune ability are closely related. A large number of dairy cows postpartum lactation, many nutrient loss, resulting in reduced immunity. The milk Gland tissue of the external invasion of pathogenic defenses reduce pathogenic microorganism infection caused by mastitis. Studies show that selenium deficiency promote early lactation mastitis, increasing dietary selenium intake can significantly improve the phagocytic activity and reduce the occurrence of mastitis. Many clinical studies also found that bovine Staphylococcus aureus mastitis prevalence rate and selenium content in milk in the present linear correlation of dietary selenium can reduce breast infection significantly reduced the incidence of mastitis, but the mechanism is still unclear. In this study, selenium on breast local immune regulation as a starting point to explore the effect of Staphylococcus aureus mastitis and preliminary analysis of its mechanism firstly. Mice were fed with different selenium concentration in grain, and then establish S.aureus mastitis model, simultaneous separation of mammary epithelial cells was studied. Results the cash s. Bacterial infection leads to breast tissue structure was destroyed, a large number of mammary epithelial cell shedding death. Selenium fed mice inflammatory injury is the most serious, and the selenium fed mice relatively mild inflammatory changes. On inflammatory factor analysis found that selenium deficiency induced by promoting breast tissue IL-1 beta, Staphylococcus aureus infection of TNF- alpha, IL-6,12 IFN-, gamma, NO secretion of pro-inflammatory factor, inhibition of PPAR- gamma, IL-10 secretion of anti-inflammatory cytokines. Selenium can reduce feeding proinflammatory cytokine secretion, increase anti-inflammatory cytokine secretion. We isolated mammary epithelial cells by different concentration of selenium containing medium incubationa549, give corresponding research on Staphylococcus aureus was obtained after stimulation, and study results are consistent. We further for S. aureus pathogen recognition receptor TLR2 and its downstream inflammatory signaling pathways were analyzed. The results showed that selenium on NF- kappa B and MAPKs signaling pathway molecules gene expression level No effect, but the addition of selenium can reduce the excessive expression of TLR2 inflammation, inhibit the expression of NF- K B and MAPK signaling pathways start, reduce inflammatory injury. Over expression of selenium deficiency increased TLR2, increased inflammatory response. Staphylococcus aureus mastitis showed inflammation, also can cause apoptosis of breast epithelial cells and destroy breast tissue structure. In order to analysis the effects of selenium, through in vivo and in vitro experiments on cell apoptosis and injury were analyzed. The results showed that selenium has a good regulatory role on breast tissue cell apoptosis induced by S.aureus. Selenium deficiency increased the phosphorylation of p53, improve the BAX/BCL-2 value, promote Caspase-3,6,7 protein cleavage, accelerated apoptosis process induced by Staphylococcus aureus start. Selenium can inhibit cell apoptosis program exerts a protective effect on breast tissue. As everyone knows, through its Selenium Selenoprotein A variety of biological functions. In many selenoproteins have been identified in selenoprotein S (Sel S) is closely related to inflammation, for the further study of the regulatory mechanism of selenium on Staphylococcus aureus mastitis, we use Sel S as the target, through in-depth study of RNA interference technology. Studies have shown selenium by influencing the expression of S protein in selenoprotein phosphorylation regulation of NF- kappa B and MAPK signaling pathways molecules, secretion of inflammatory cytokines. Selenium can regulate the phosphorylation of P53 by Bax Bcl-2, selenoprotein S, protein expression and Caspase-3,6,7 apoptotic factor protein cleavage of Staphylococcus aureus mastitis occurrence regulation. In conclusion, selenium of Staphylococcus aureus mastitis occurrence plays an important role in the regulation of.Sel S plays an important role. This study is to clarify the pathogenesis of staphylococcal mastitis, search The aim is to provide new targets and ideas for the prevention and treatment of dairy cow mastitis, and to enrich the biological function of selenium, so as to provide theoretical basis and help for the development of nutrition and immunology in animal diseases.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.23

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