本文關(guān)鍵詞：MKK3激酶在機(jī)體抵御金黃色葡萄球菌感染中的功能研究　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： MKK3激酶 金黃色葡萄球菌 皮膚感染 巨噬細(xì)胞 炎性體

【摘要】：金黃色葡萄球菌是引起群體感染一種最常見革蘭陽性人獸共患病原菌,也是引起全球范圍內(nèi)感染的重要致病菌,主要存在于動物的皮膚和呼吸道黏膜等部位。金黃色葡萄球菌作為條件機(jī)會性致病菌,當(dāng)機(jī)體免疫功能低下或皮膚黏膜出現(xiàn)破損時,細(xì)菌可突破皮膚及黏膜屏障,導(dǎo)致宿主皮膚、肺部等部位感染性疾病發(fā)生,重度感染可因敗血癥、膿毒癥等導(dǎo)致宿主死亡。近年來,由于臨床上抗生素的大量使用,導(dǎo)致多重耐藥性金黃色葡萄球菌菌株產(chǎn)生,其致病性不斷提高,病死率逐年增加。在機(jī)體感染和抗感染過程中,固有免疫系統(tǒng)作為機(jī)體抵御病原微生物感染的第一道防線,擔(dān)負(fù)著控制感染擴(kuò)散和炎癥反應(yīng)等重要角色。MAPKs絲裂原活化蛋白激酶家族是生物體內(nèi)重要的信號轉(zhuǎn)導(dǎo)系統(tǒng),MKK3基因編碼蛋白為絲裂原活化蛋白激酶激酶3,屬絲裂原活化蛋白激酶家族中的重要成員。大量研究表明MKK3信號在天然免疫調(diào)節(jié)、炎癥反應(yīng)以及細(xì)胞因子產(chǎn)生等過程中發(fā)揮關(guān)鍵性作用,然而MKK3在金黃色葡萄球菌感染中的生物學(xué)作用仍尚未澄清。為了探究和闡明MKK3在金黃色葡萄球菌感染過程中的作用,本實(shí)驗(yàn)利用WT小鼠和MKK3基因缺失小鼠,通過皮下注射一定感染劑量的金黃色葡萄球菌構(gòu)建皮膚感染動物模型,探究MKK3在金黃色葡萄球菌皮膚感染中的作用。實(shí)驗(yàn)結(jié)果表明,與WT小鼠相比,MKK3基因缺失后,小鼠皮膚膿腫潰瘍面積顯著增大,皮膚組織微觀病理變化更為嚴(yán)重,皮膚真皮層炎性細(xì)胞浸潤及水腫等現(xiàn)象更為明顯,皮膚組織中的炎性介質(zhì)表達(dá)、細(xì)胞因子及趨化因子分泌量均顯著增加(p0.05),但在感染早期小鼠皮膚感染部位細(xì)菌定植量差異不顯著(p0.05)。以上結(jié)果說明,小鼠MKK3基因缺失后表現(xiàn)對金黃色葡萄球菌的易感性增強(qiáng),炎性反應(yīng)增加,即在宿主抵御金黃色葡萄球菌皮膚感染過程中MKK3發(fā)揮著重要作用。隨后對MKK3參與調(diào)節(jié)炎性反應(yīng)機(jī)制進(jìn)行了初步探討。通過TUNEL染色分析皮膚組織中細(xì)胞凋亡情況;免疫熒光分析皮膚組織中PCNA表達(dá);免疫組化分析皮膚組織中p-MKK3在表達(dá)。結(jié)果表明,未感染金黃色葡萄球菌時,WT小鼠與MKK3基因缺失小鼠皮膚組織中凋亡及增殖的細(xì)胞數(shù)量均較少。而感染金黃色葡萄球菌后,同WT小鼠相比,MKK3基因缺失后,組織中細(xì)胞凋亡主要分布在皮膚淺表層,且凋亡數(shù)量差異不顯著(p0.05);而皮膚組織中PCNA表達(dá)量則顯著增加(p0.0001);p-MKK3主要表達(dá)于炎性細(xì)胞中。近年來研究表明,細(xì)胞因子IL-1β在金黃色葡萄球菌感染過程中皮膚膿腫形成和炎性細(xì)胞趨化過程中發(fā)揮重要作用,而該細(xì)胞因子來源主要依賴于炎性體活化產(chǎn)生。體外實(shí)驗(yàn)以原代小鼠腹腔巨噬細(xì)胞為研究模型,分析金黃色葡萄球菌感染過程中,MKK3參與炎性體活化的調(diào)控作用。免疫蛋白印跡結(jié)果表明,與WT處理組相比,巨噬細(xì)胞缺失MKK3基因后Caspase-1、IL-1β、ASC蛋白表達(dá)量均顯著增強(qiáng),且該過程主要依賴于p-JNK信號。為進(jìn)一步揭示MKK3在宿主抵御金葡菌感染皮膚過程中所發(fā)揮作用。通過選取金葡菌感染小鼠皮膚較長時間點(diǎn),對皮膚組織中金葡菌的定植情況進(jìn)行分析。結(jié)果表明,在金葡菌感染第12天和第14天,與WT感染組相比,MKK3基因缺失后皮膚組織中細(xì)菌定植量增加顯著(p0.05)。說明在金葡菌感染晚期,MKK3基因缺失后不利于巨噬細(xì)胞清除病原菌,對巨噬細(xì)胞的正常殺菌功能產(chǎn)生影響。綜上所述,本研究表明MKK3激酶可通過抑制JNK磷酸化,抑制巨噬細(xì)胞炎性體活化,從而抑制金黃色葡萄球菌感染引起強(qiáng)烈的炎性反應(yīng),促進(jìn)巨噬細(xì)胞的殺菌功能,對宿主起保護(hù)作用。
[Abstract]:Staphylococcus aureus is one of the most common infection group caused by Gram positive zoonotic pathogens, is also an important pathogen worldwide infection, skin and respiratory tract mucosa mainly exists in animal. Staphylococcus aureus as opportunistic pathogenic bacteria, when the immune dysfunction or skin mucosa damage when bacteria can break the skin and mucosal barrier, leading to host the skin, lungs and other parts of infectious diseases, severe infections due to sepsis, sepsis and other causes of host death. In recent years, due to the extensive use of antibiotics, resulting in multiple drug resistant Staphylococcus aureus strains, the pathogenicity of increasing mortality the increasing rate of infection and anti infection. In the process, the innate immune system as the first line of defense against infection of pathogenic microorganisms, responsible for infection control The diffusion and the inflammatory reaction of the important role of.MAPKs mitogen activated protein kinase family is an important signal transduction system in vivo, MKK3 gene encoding a protein of mitogen activated protein kinase kinase 3 is an important member of mitogen activated protein kinase family. A large number of studies show that MKK3 signaling regulates the innate immunity, play a key role in the inflammatory reaction process and cytokines, but the biological function of MKK3 in Staphylococcus aureus infection is still not clarified. In order to explore and clarify the role of MKK3 in Staphylococcus aureus infection, this experiment using WT mice and MKK3 knockout mice by subcutaneous injection of a certain dose of infection, Staphylococcus aureus, animal model of skin to explore the role of MKK3 in skin infection, infection of Staphylococcus aureus. Experimental results show that, compared with WT mice, MKK3 Due to lack of mouse skin abscess, ulcer area was significantly increased and the microscopic pathological changes of skin tissue is more serious, the dermal inflammatory cell infiltration and edema is more obvious, the expression of inflammatory mediators in skin tissue, cytokines and chemokines secretion were significantly increased (P0.05), but in the early stage of infection in mice the skin infection of bacterial colonization were not significant (P0.05). These results indicated that MKK3 mutant mice showed susceptibility of Staphylococcus aureus increased, increased inflammatory reaction, namely against Staphylococcus aureus skin infection in the process of MKK3 plays an important role in the host. Then the MKK3 is involved in the regulation of inflammatory mechanism were studied. Analysis of cell apoptosis in skin tissue by TUNEL staining; immunofluorescence analysis of the expression of PCNA in skin tissue; immunohistochemical analysis of skin tissue in table p-MKK3 Not up. The results showed that Staphylococcus aureus infection, the number of cell apoptosis and proliferation of skin tissue of mice WT mice and MKK3 gene deletion in less. MRSA, compared with WT mice, MKK3 gene deletion and apoptosis in tissue mainly distributed in the superficial layer of the skin, and the number of apoptosis the difference was not significant (P0.05); and the expression of PCNA in skin tissue were significantly increased (P0.0001); p-MKK3 was mainly expressed in inflammatory cells. Recent studies have shown that beta cell factor IL-1 in Staphylococcus aureus infection and inflammatory cell migration plays an important role in the process of the formation of skin abscess in the process, and the main source of cytokine dependent on inflammasome activation. In vitro in primary peritoneal macrophages of mice model for the study, analysis of Staphylococcus aureus infection process, MKK3 is involved in the regulation role of inflammasome activation free. And Western blot results showed that, compared with WT group, macrophage MKK3 gene deletion Caspase-1, IL-1 beta, ASC protein expression were significantly increased, and the process mainly depends on the p-JNK signal. In order to further reveal the MKK3 play a role in the host against the skin of Staphylococcus aureus infection. By selecting the skin for a long time. S. aureus infected mice, analyze the colonization of Staphylococcus aureus on skin tissue. The results showed that in twelfth days and fourteenth days of Staphylococcus aureus infection, compared with WT infection group, the bacterial colonization amount of skin tissue after gene deletion in MKK3 increased significantly (P0.05). In the late Staphylococcus aureus infection, MKK3 gene after the loss is not conducive to the removal of macrophages pathogens, which affects the normal bactericidal function of macrophage. In summary, this study indicates that MKK3 kinase can inhibit JNK phosphorylation, inhibit macrophage inflammatory activation, thereby inhibiting Staphylococcus aureus infection causes a strong inflammatory response, promotes the bactericidal function of macrophages and protects the host.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.611

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1 李坤t@;MKK3激酶在機(jī)體抵御金黃色葡萄球菌感染中的功能研究[D];吉林大學(xué);2017年

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本文編號：1357990