MKK3激酶在机体抵御金黄色葡萄球菌感染中的功能研究

发布时间：2017-12-31 03:45

本文关键词：MKK3激酶在机体抵御金黄色葡萄球菌感染中的功能研究　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：金黄色葡萄球菌是引起群体感染一种最常见革兰阳性人兽共患病原菌,也是引起全球范围内感染的重要致病菌,主要存在于动物的皮肤和呼吸道黏膜等部位。金黄色葡萄球菌作为条件机会性致病菌,当机体免疫功能低下或皮肤黏膜出现破损时,细菌可突破皮肤及黏膜屏障,导致宿主皮肤、肺部等部位感染性疾病发生,重度感染可因败血症、脓毒症等导致宿主死亡。近年来,由于临床上抗生素的大量使用,导致多重耐药性金黄色葡萄球菌菌株产生,其致病性不断提高,病死率逐年增加。在机体感染和抗感染过程中,固有免疫系统作为机体抵御病原微生物感染的第一道防线,担负着控制感染扩散和炎症反应等重要角色。MAPKs丝裂原活化蛋白激酶家族是生物体内重要的信号转导系统,MKK3基因编码蛋白为丝裂原活化蛋白激酶激酶3,属丝裂原活化蛋白激酶家族中的重要成员。大量研究表明MKK3信号在天然免疫调节、炎症反应以及细胞因子产生等过程中发挥关键性作用,然而MKK3在金黄色葡萄球菌感染中的生物学作用仍尚未澄清。为了探究和阐明MKK3在金黄色葡萄球菌感染过程中的作用,本实验利用WT小鼠和MKK3基因缺失小鼠,通过皮下注射一定感染剂量的金黄色葡萄球菌构建皮肤感染动物模型,探究MKK3在金黄色葡萄球菌皮肤感染中的作用。实验结果表明,与WT小鼠相比,MKK3基因缺失后,小鼠皮肤脓肿溃疡面积显著增大,皮肤组织微观病理变化更为严重,皮肤真皮层炎性细胞浸润及水肿等现象更为明显,皮肤组织中的炎性介质表达、细胞因子及趋化因子分泌量均显著增加(p0.05),但在感染早期小鼠皮肤感染部位细菌定植量差异不显著(p0.05)。以上结果说明,小鼠MKK3基因缺失后表现对金黄色葡萄球菌的易感性增强,炎性反应增加,即在宿主抵御金黄色葡萄球菌皮肤感染过程中MKK3发挥着重要作用。随后对MKK3参与调节炎性反应机制进行了初步探讨。通过TUNEL染色分析皮肤组织中细胞凋亡情况;免疫荧光分析皮肤组织中PCNA表达;免疫组化分析皮肤组织中p-MKK3在表达。结果表明,未感染金黄色葡萄球菌时,WT小鼠与MKK3基因缺失小鼠皮肤组织中凋亡及增殖的细胞数量均较少。而感染金黄色葡萄球菌后,同WT小鼠相比,MKK3基因缺失后,组织中细胞凋亡主要分布在皮肤浅表层,且凋亡数量差异不显著(p0.05);而皮肤组织中PCNA表达量则显著增加(p0.0001);p-MKK3主要表达于炎性细胞中。近年来研究表明,细胞因子IL-1β在金黄色葡萄球菌感染过程中皮肤脓肿形成和炎性细胞趋化过程中发挥重要作用,而该细胞因子来源主要依赖于炎性体活化产生。体外实验以原代小鼠腹腔巨噬细胞为研究模型,分析金黄色葡萄球菌感染过程中,MKK3参与炎性体活化的调控作用。免疫蛋白印迹结果表明,与WT处理组相比,巨噬细胞缺失MKK3基因后Caspase-1、IL-1β、ASC蛋白表达量均显著增强,且该过程主要依赖于p-JNK信号。为进一步揭示MKK3在宿主抵御金葡菌感染皮肤过程中所发挥作用。通过选取金葡菌感染小鼠皮肤较长时间点,对皮肤组织中金葡菌的定植情况进行分析。结果表明,在金葡菌感染第12天和第14天,与WT感染组相比,MKK3基因缺失后皮肤组织中细菌定植量增加显著(p0.05)。说明在金葡菌感染晚期,MKK3基因缺失后不利于巨噬细胞清除病原菌,对巨噬细胞的正常杀菌功能产生影响。综上所述,本研究表明MKK3激酶可通过抑制JNK磷酸化,抑制巨噬细胞炎性体活化,从而抑制金黄色葡萄球菌感染引起强烈的炎性反应,促进巨噬细胞的杀菌功能,对宿主起保护作用。
[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.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.611

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