發(fā)布時(shí)間：2018-01-08 06:17

  本文關(guān)鍵詞：硒對(duì)金黃色葡萄球菌性乳腺炎發(fā)生的影響及調(diào)控機(jī)制研究　出處：《吉林大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 硒 乳腺炎 金黃色葡萄球菌 炎癥 硒蛋白S

【摘要】：乳腺炎是嚴(yán)重危害奶牛健康的重要疾病。該病導(dǎo)致患牛泌乳量減少、奶牛淘汰率增加,同時(shí)引發(fā)相關(guān)食品安全問(wèn)題。奶牛乳腺炎的發(fā)生與許多因素有關(guān),其中病原感染是奶牛乳腺炎發(fā)生的主要原因。諸多報(bào)道顯示多種微生物均可引發(fā)該病,包括病毒、寄生蟲(chóng)、支原體、真菌及細(xì)菌等等。金黃色葡萄球菌(金葡菌)是誘發(fā)奶牛乳腺炎的主要病原。然而,我們臨床調(diào)查發(fā)現(xiàn)并不是金葡菌入侵的所有奶牛都發(fā)生乳腺炎。這些說(shuō)明該病的發(fā)生與動(dòng)物機(jī)體的自身免疫狀態(tài)密切相關(guān)。眾所周知,營(yíng)養(yǎng)狀態(tài)與哺乳動(dòng)物的免疫能力密切相關(guān)。奶牛產(chǎn)后大量泌乳,眾多營(yíng)養(yǎng)物質(zhì)損失,導(dǎo)致機(jī)體免疫力降低。此時(shí)乳腺組織對(duì)外界入侵病原防御能力降低,致病微生物感染引發(fā)乳腺炎。研究顯示,缺硒促進(jìn)泌乳早期乳腺炎的發(fā)生,增加日糧硒攝入可顯著提高細(xì)胞吞噬活性并降低乳腺炎發(fā)生。大量的臨床研究還發(fā)現(xiàn),奶牛金葡菌性乳腺炎患病率與牛乳中硒含量呈現(xiàn)線性相關(guān)。日糧中適當(dāng)補(bǔ)硒可減少乳腺病原菌感染,顯著降低乳腺炎發(fā)病率,但其中的作用機(jī)制目前還不清楚。本研究以硒對(duì)乳腺局部免疫調(diào)控作用為切入點(diǎn),探究其對(duì)金葡菌性乳腺炎發(fā)生影響并初步分析其作用機(jī)制。研究首先飼喂小鼠不同含硒濃度的鼠糧,然后建立金葡菌乳腺炎模型,同時(shí)分離乳腺上皮細(xì)胞進(jìn)行相關(guān)研究。結(jié)果發(fā)現(xiàn)金葡菌感染導(dǎo)致乳腺組織結(jié)構(gòu)被破壞,大量乳腺上皮細(xì)胞脫落死亡。缺硒飼喂小鼠炎性損傷最為嚴(yán)重,而富硒飼喂小鼠炎癥變化相對(duì)輕微。對(duì)炎性因子進(jìn)行分析發(fā)現(xiàn),缺硒促進(jìn)乳腺組織因金葡菌感染導(dǎo)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。富硒飼喂可相對(duì)降低促炎因子分泌,增加抗炎因子分泌。我們分離乳腺上皮細(xì)胞,通過(guò)不同濃度含硒培養(yǎng)基孵育,給予金葡菌刺激后進(jìn)行相應(yīng)的研究,獲得了與組織研究相一致的結(jié)果。我們進(jìn)一步對(duì)金葡菌的主要識(shí)別受體TLR2及其下游炎癥調(diào)控信號(hào)通路進(jìn)行分析。結(jié)果發(fā)現(xiàn),硒對(duì)NF-κB和MAPKs信號(hào)通路各分子基因表達(dá)水平?jīng)]有影響,但添加硒可降低炎癥發(fā)生后TLR2的過(guò)度表達(dá),抑制其下游NF-κB及MAPK炎癥信號(hào)通路的啟動(dòng),降低炎性損傷。缺硒則會(huì)增加TLR2的過(guò)度表達(dá),加重炎癥反應(yīng)。金葡菌性乳腺炎除表現(xiàn)炎癥外,還會(huì)引發(fā)乳腺上皮細(xì)胞凋亡而破壞乳腺組織結(jié)構(gòu)。為了全面解析硒的作用,我們通過(guò)在體及體外實(shí)驗(yàn)對(duì)細(xì)胞凋亡及損傷情況進(jìn)行分析。結(jié)果顯示硒對(duì)金葡菌誘導(dǎo)的乳腺組織細(xì)胞凋亡具有很好的調(diào)節(jié)作用。缺硒增加p53磷酸化,提高BAX/BCL-2值,促進(jìn)Caspase-3、6、7蛋白裂解,加速金葡菌誘導(dǎo)的細(xì)胞凋亡程序啟動(dòng)。補(bǔ)硒可以抑制細(xì)胞凋亡程序啟動(dòng),從而對(duì)乳腺組織發(fā)揮保護(hù)作用。眾所周知,硒通過(guò)硒蛋白發(fā)揮其各種生物學(xué)功能。在諸多硒蛋白中已經(jīng)證實(shí)硒蛋白S(Sel S)與炎癥發(fā)生密切相關(guān),為進(jìn)一步深入研究硒對(duì)金葡菌乳腺炎發(fā)生的調(diào)控機(jī)制,我們以Sel S為靶點(diǎn),通過(guò)RNA干擾技術(shù)進(jìn)行深入研究。研究顯示硒通過(guò)影響硒蛋白S的表達(dá)調(diào)控NF-κB和MAPK炎癥信號(hào)通路各分子的蛋白磷酸化水平,調(diào)節(jié)炎癥相關(guān)因子的分泌。硒可通過(guò)硒蛋白S可調(diào)控P53磷酸化水平,Bax,Bcl-2蛋白表達(dá)及Caspase-3、6、7凋亡執(zhí)行因子蛋白裂解,從而對(duì)金葡菌乳腺炎的發(fā)生發(fā)揮調(diào)控作用。綜上表明,硒對(duì)金葡菌性乳腺炎的發(fā)生發(fā)揮著重要調(diào)控作用。Sel S在這個(gè)調(diào)控過(guò)程中發(fā)揮著重要的作用。本研究為闡明金葡菌性乳腺炎的發(fā)病機(jī)制,尋求奶牛乳腺炎防治藥物及有效措施提供新的靶點(diǎn)和思路。同時(shí)豐富硒的生物學(xué)作用,為營(yíng)養(yǎng)免疫學(xué)在動(dòng)物疾病方面的發(fā)展提供理論基礎(chǔ)和幫助。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S858.23

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