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临床感染持留菌的筛选及其感染免疫机制研究

发布时间:2019-06-25 15:20
【摘要】:持留菌(Persisters)是存在于某个细菌群体中的表型异化小亚群。可以耐受致死浓度的抗菌药物,国内外学者认为其与慢性感染的反复发作及细菌的生物被膜感染等密切相关。肺炎克雷伯菌是重要的医院和社区获得性革兰阴性机会致病菌,可导致患者发生尿路感染、肺炎、菌血症及肝脓肿等多种疾病。本课题针对重要的临床耐药问题,利用实验室感染病原样本库,从建立微生物快速诊断技术和临床样本持留菌筛选入手,研究顽固性难治性感染相关的持留菌的感染免疫调控机制。通过观察持留菌流行病学特征和持留特性,从感染免疫角度阐述单核巨噬细胞与持留菌之间的相互作用,解释免疫逃避机制,为免疫干预治疗提供理论依据。第一部分临床细菌感染现状分析及分子诊断检验本部分研究对临床细菌感染现状进行分析,对医院相关感染进行流行病学调查;分析耐药的分子基础,形成分子诊断方案,为临床感染控制提供预警。建立了基于多重PCR体系的主要耐药菌快速鉴定和耐药机制检测的分子生物学方法,可以很好的作为传统微生物检验方法的补充,有利于对临床耐药菌株进行耐药机制的系统分析和调查,更好地为感染防控提供依据。第二部分肺炎克雷伯杆菌持留株的筛选及特性研究本部分研究利用本院建立的菌株样本库,对临床菌株进行了持留菌的筛选并对其持留特性和耐药性进行了研究。利用生长曲线法,共筛选获得51株持留菌,其中26株(48.9%)为肺炎克雷伯杆菌。26株肺炎克雷伯杆菌持留株全部产ESBLs,对氨苄西林100%耐药。对哌拉西林/他唑巴坦等耐药率均超过75%,对环丙沙星的耐药率为60.8%。随环丙沙星浓度升高,持留菌比例下降,甚至在50MIC浓度时可以实现对持留菌的杀灭。这一现象提示我们,不能通过判读临床常规药敏试验结果来区分持留菌,持留菌由于缺乏明确的特异标志物而难以严格区分和鉴定,这是持留菌研究中的一个难点和值得挖掘的研究切入点;此外,虽然体外实验中抗生素浓度达到一定值时可以杀灭持留菌,但实际上体内不可能达到这样的抗生素浓度,靠加大抗生素剂量的方法治疗持留菌感染行不通,因而,拟进一步探讨持留菌的持留机制或者其感染免疫机制,以期为解决这一临床问题提供理论依据。第三部分肺炎克雷伯菌持留株-单核巨噬细胞间相互作用及机制研究针对前期筛选出的临床持留菌株,从感染免疫角度阐述单核巨噬细胞与持留菌之间的相互作用。首先通过体外实验发现单核细胞对持留菌的吞噬作用降低,进一步通过芯片检测、生物信息学分析和实验验证来确定单核巨噬细胞中免疫相关基因的表达及其效应信号通路。发现持留菌感染单核巨噬细胞,可以诱导SOCS-1蛋白在感染初期即大量表达,且部分依赖于TLR4信号通路;SOCS-1蛋白可以下调MAL的水平进而影响TLR4下游信号通路的活化,降低TNF-α、IL-6、IL-1β等炎症因子的分泌。证明肺炎克雷伯菌持留株感染可以通过诱导产生的SOCS-1负调控炎症因子的表达,从而逃逸宿主免疫攻击。本课题从感染免疫角度阐述单核巨噬细胞与持留菌之间的相互作用,探讨了持留菌的免疫逃避机制,为免疫干预治疗提供了理论依据。
[Abstract]:Persorters are a small subset of the phenotype that is present in a certain bacterial population. The anti-bacterial drug with lethal concentration can be tolerated, and the domestic and foreign scholars believe that it is closely related to the repeated attack of the chronic infection and the biological membrane infection of the bacteria. Klebsiella pneumoniae is an important hospital and community-acquired Gram-negative opportunistic pathogen, which can lead to a variety of diseases such as urinary tract infection, pneumonia, bacteremia, and liver abscess. Aiming at the important clinical drug-resistance problem, using the laboratory-infected pathogen sample library, the paper starts with the establishment of the micro-organism rapid diagnosis technology and the screening of the clinical sample holding-keeping bacteria, and studies the infection-immune regulation mechanism of the bacteria-retaining bacteria associated with the intractable intractable infection. In order to provide the theoretical basis for the treatment of immune intervention, by observing the epidemiological characteristics and the holding characteristics of the retained bacteria, the interaction between the mononuclear macrophages and the retaining bacteria is described from the angle of infection, and the immune escape mechanism is explained. The present situation of clinical bacterial infection in the first part and the analysis of the present part of the molecular diagnosis and examination of the present part of the clinical bacteria infection are analyzed, and the epidemiological investigation on the infection of the hospital is carried out. The molecular basis of the drug resistance is analyzed, and the molecular diagnosis scheme is formed, and the early warning is provided for the control of clinical infection. A molecular biological method based on the rapid identification of the main drug-resistant bacteria and the detection of the drug resistance mechanism based on the multiple PCR system is established, which can be used as a supplement to the traditional microbial test method, and is beneficial to the system analysis and investigation of the drug-resistant mechanism of the clinical drug-resistant strain, And provides a basis for better prevention and control of infection. The screening and characterization of the second part of Klebsiella pneumoniae (Klebsiella pneumoniae) were studied in this part. The strain samples library established in our hospital were used to screen and study the retention characteristics and drug resistance of the strains. The results showed that 26 strains (48.9%) were Klebsiella pneumoniae and 26 strains (48.9%) were Klebsiella pneumoniae.26 strains of Klebsiella pneumoniae were all ESBLs, which were resistant to 100% of methicillin. The drug resistance rate was over 75%, and the resistance rate of ciprofloxacin to ciprofloxacin was 60.8%. With the increase of the concentration of ciprofloxacin, the proportion of the retained bacteria is decreased, and the killing of the retaining bacteria can be realized even at the concentration of 50 MIC. This phenomenon suggests that we can not distinguish the remaining bacteria from the results of the clinical routine drug sensitivity test, which is difficult to distinguish and identify by the lack of specific specific markers, which is a difficult and worthwhile research entry point in the study of retained bacteria; in addition, Although the concentration of the antibiotic in the in vitro experiment reaches a certain value, the stay-keeping bacteria can be killed, It is proposed to study the retention mechanism or the immune mechanism of the retained bacteria in order to provide a theoretical basis for the solution of this clinical problem. The interaction and mechanism of the third part of klebsiella pneumoniae (klebsiella pneumoniae)-mononuclear macrophages (kleklebsiella) on the pre-screened clinical retention strains, the interaction between the mononuclear macrophages and the retaining bacteria was described from the angle of infection. First, by in vitro experiments, the phagocytosis of the mononuclear macrophages was reduced, and the expression of the immune-related genes in the mononuclear macrophages and their effect signal pathways were further determined by chip detection, bioinformatics analysis and experimental verification. It is found that the SOCS-1 protein can be expressed in the early stage of infection, and the part of the SOCS-1 protein is dependent on the TLR4 signal pathway. The SOCS-1 protein can down-regulate the level of the MAL and then influence the activation of the signal pathway downstream of the TLR4, and the secretion of the inflammatory factors such as TNF-1, IL-6, IL-1 and the like can be reduced. It is proved that the infection of the strain of Klebsiella pneumoniae can control the expression of the inflammatory factor by inducing the generated SOCS-1 to escape the host immune attack. In this paper, the interaction between the mononuclear macrophages and the retaining bacteria is described from the angle of infection, and the immune escape mechanism of the retaining bacteria is discussed, which provides a theoretical basis for the treatment of immune intervention.
【学位授予单位】:第二军医大学
【学位级别】:博士
【学位授予年份】:2016
【分类号】:R515

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