类鼻疽菌抑制巨噬细胞自噬逃逸清除的分子机制和流行病学研究

发布时间：2018-06-05 20:23

本文选题：类鼻疽 + 类鼻疽菌　；参考：《第三军医大学》2016年博士论文

【摘要】：类鼻疽伯克霍尔德菌(简称类鼻疽菌)属兼性胞内菌,作为人类鼻疽病的病原体,是一种环境寄生的机会致病菌,广泛分布于东南亚和澳洲北部。我国海南、广东和广西等地是类鼻疽的疫源地。2016年的第1期Nature Mirobiology杂志对全球类鼻疽菌流行扩散形势和感染风险进行预测分析,估算2015年在全球范围内类鼻疽感染病例超过16万人,死亡人数约9万人。鉴于类鼻疽的严重致病性,美国CDC早在2006年就将类鼻疽菌列为I类病原体严加防范。90年代,我国学者宋阳等已经报道过类鼻疽菌在海南的地理分布,当地疾控中心也曾进行过类鼻疽菌的环境监测,但可查资料仍然很少,类鼻疽的扩散之势可能早已远超我们估计。多位点序列分型(MLST)是一种基于核酸序列(七个管家基因)测定的细菌分型方法,也可用于分析不同ST型之间的系统发育关系以及与疾病的联系。早在2003年MLST就被应用到类鼻疽菌的分子流行病学研究中,它操作简单,分辨力高,更便于国际同行进行数据共享。目前,我国还没有类鼻疽菌MLST的相关文章报道,分析我国类鼻疽的流行形势和类鼻疽菌的变化趋势显得日益重要。因此,我们随机选取了跨越11年的102株临床类鼻疽菌株进行了MLST分型,借此分析发现近年我国类鼻疽菌的变异规律和扩散趋势,进一步完善我国类鼻疽菌株基因信息数据库,为我国类鼻疽的溯源和预警提供参考。2012年Wiersinga WJ在新英格兰杂志报道了类鼻疽的全球流行趋势,估算其发病率达50/100000,引起了国际同行对于类鼻疽研究的高度关注。在中国,类鼻疽的研究并未引起足够的重视有多方面的原因,其中,首要的原因是诊断困难,导致类鼻疽的漏诊率极高。其次是类鼻疽的临床表现多样缺乏特征,易误诊为其他感染性疾病。即使在海南当地,很多医生仍然对类鼻疽比较陌生,更不用说内地的医疗工作者了。为此,我们基于海南当地三所最大医院的数据库,收集了跨越11年共计170例类鼻疽病例的临床资料,汇总分析了我国类鼻疽感染风险、流行趋势、地理分布、季节分布和临床特征,为我国类鼻疽的临床诊断和调查分析提供借鉴和参考。临床类鼻疽治疗中最难控制的问题即是感染的慢性化和复发。即使在规范抗生素用药情况下,复发率仍达到20%。已有文献报道,类鼻疽菌可以侵入几乎所有的宿主细胞,在其中寄居和生存,并导致宿主细胞融合实现病原菌自身的增殖和扩散。而这可能就是导致类鼻疽感染慢性化和易复发的重要原因。自噬是清除胞内病原体的重要途径之一,自噬体捕获病原体,通过与溶酶体结合,形成成熟的自噬溶酶体降解细菌从而达到抵抗胞内菌入侵和感染的目的。但是有些病原体已经进化出能够抵抗或者逃避自噬清除的机制,而类鼻疽与宿主自噬之间的关系以及它是如何抵制宿主细胞自噬清除的机制还有待进一步阐明。我们前期的研究已经发现类鼻疽可以通过诱导某些特殊mirna来抑制人肺上皮细胞的自噬水平,抑制自噬体的形成从而实现在胞内的生存和增殖。基于对类鼻疽菌感染的小鼠巨噬细胞raw264.7的基因芯片分析,我们发现,类鼻疽菌抑制小鼠细胞的自噬消化并不是通过抑制自噬体形成,而是通过影响自噬体与溶酶体的融合、抑制自噬溶酶体的成熟来实现的,而且这一抑制过程也是由类鼻疽菌诱导的特异性mirna所介导。这项研究表明,自噬,尤其是完整的自噬对于宿主细胞对抗胞内菌感染是非常重要的。同时,前期研究结果也提示我们,微生物诱导的mirna在影响宿主细胞信号通路中具有重要作用,可能与许多的微生物感染进程紧密相关,也暗示我们利用小rna干扰策略或许能够给对抗微生物感染提供不一样的治疗思路。方法1.中国海南类鼻疽-回顾性分析1.1基于海南当地三所最大医院的数据库,收集跨越11年共计170例类鼻疽病例的临床资料;1.2所有病例均需临床和实验室确认类鼻疽诊断;1.3汇总分析我国类鼻疽感染风险、流行趋势、地理分布、季节分布和临床特征。2.类鼻疽菌的mlst分析2.1对102株临床分离的类鼻疽菌株首先进行生化与16srdnapcr鉴定;2.2对类鼻疽菌ace,ghmd,gltb,lipa,lepa,nark,ndh七个管家基因进行扩增和测序分析;2.3在线分析测序结果,确定st型,上传数据,在线对本次研究菌株序列或者综合类鼻疽菌mlst数据库序列进行e-burst分析,upgma方法构建系统进化树对收集的菌株进行溯源分析等;2.4运用mlst及16srdna分型等方法对镇江1例输入性类鼻疽病例进行实验室确诊和溯源分析。3.类鼻疽菌抑制巨噬细胞自噬研究3.1类鼻疽菌感染raw细胞模型和不同感染时相条件下的表达谱芯片分析;3.2小rna干扰或者真核过表达,用于分析自噬信号通路关键蛋白在类鼻疽的胞内增殖作用;3.3激光共聚焦、高分辨显微镜和透射电镜用于观察胞内lc3b转化,细胞融合,胞内菌生存情况,自噬体以及自噬溶酶体的形成;3.4cfu平板计数用于定量细胞内类鼻疽菌的增殖情况;3.5qrt-pcr用于定量mrna水平,而westernblot用于反映自噬分子蛋白水平;3.6荧光素酶实验用于mir-146a靶基因鉴定试验;3.7数据分析:logsticregression用于危险因素相关性分析;e-burst用于mlst分型数据分析;clcgenomicsworkbench用于序列分析和引物设计;graphpadprism5.0统计作图;zen2012分析共聚焦数据;bio-radcfxmanager3.0用于定量pcr结果分析;adobeillustratorcs3进行图片整合;student’st检验用于检测两样本之间差异,*p0.05有统计学意义。结果1.中国海南类鼻疽临床回顾性研究1.1中国类鼻疽病例呈现增长态势;1.2中国类鼻疽临床表现有自身特点,主要以肺炎和败血症为主,脏器脓肿、儿童感染和颅内感染较少见;1.3类鼻疽的感染与机体免疫因素相关,相当一部分呈现慢性感染;2.mlst分析2.1102株类鼻疽菌st型别多达41种,发现8种新的st型,均已提交至数据库;2.2st-46,st-50,st-55,st-58,st-70和st-1095是102株类鼻疽菌中的主要st型,用于mlst分型的管家基因中gmhd表现出更多的变异特征,系统发生分析发现,中国与泰国在类鼻疽菌的进化上关系紧密,与其它疫区也可能存在交流播散;2.3准确鉴定了2014年江苏输入性类鼻疽病例并成功指导其处置。3.类鼻疽菌诱导的mir-146a抑制小鼠巨噬细胞自噬有利于其胞内的生存3.1类鼻疽菌可以诱导巨噬细胞自噬体的形成但是却能与之共处;3.2自噬体只能在类鼻疽菌感染小鼠巨噬细胞初期捕获和限制类鼻疽菌的生存和增殖;3.3类鼻疽菌虽然促进了自噬体的生存,但是却抑制了自噬流;3.4基于芯片检测和溶酶体信号通路表达谱分析,Lipa蛋白水平受抑可能是类鼻疽菌抑制宿主细胞自噬的效应靶点,而前者是细胞自噬溶酶体成熟的关键蛋白;3.5同时发现,类鼻疽菌感染后,伴随着Lipa蛋白水平受抑,miR-146a呈现上调;3.6生物信息学在线分析Lipa可能是miR-146a的靶蛋白,并经靶基因鉴定实验证实;3.7类鼻疽菌可借助mi R-146a-Lipa信号通路抑制小鼠巨噬细胞自噬,从而促进其在宿主细胞中的生存和增殖。结论本研究,一方面增强了我们对于类鼻疽的临床认识,了解了当前类鼻疽的流行形势和变异特点,完善了我国类鼻疽菌株基因信息数据库,可为临床医生尤其是疫区以外的医务工作者对类鼻疽的诊断提供借鉴和参考,同时为我国类鼻疽的溯源和预警提供指导;另一方面,通过深入探讨类鼻疽菌的自噬逃逸机制,对类鼻疽的临床棘手问题(感染慢性化和复发)提供了机理解释,提示我们微生物诱导的mi RNA在影响宿主细胞信号通路中的重要作用,为临床对抗类鼻疽菌感染治疗提供了新的思路。
[Abstract]:The genus Burke, the genus of Burke and Holder, is a facultative intracellular bacteria. As the pathogen of human nose, it is an opportunistic pathogenic bacteria, widely distributed in Southeast Asia and northern Australia. Hainan, Guangdong and Guangxi, China, are the first phase of Nature Mirobiology magazine for global nose in the epidemic area of the type of nose like disease. The spread of bacterial epidemic and the risk of infection were predicted and analyzed. In 2015, more than 160 thousand people were estimated to be infected with more than 160 thousand cases in the world, and the number of deaths was about 90 thousand. In view of the severe pathogenicity of the genus, the American CDC had been classified as I pathogen in the United States early in 2006 to prevent the case of.90, which has been reported by Chinese scholars, such as Song Yang. The geographical distribution of the bacteria in Hainan, the local CDC has also been carried out by the environmental monitoring of the Bacillus like bacteria, but the data are still very few. The spread potential of the genus is far beyond our estimate. The multibit sequence classification (MLST) is a bacterial typing method based on the nucleic acid sequence (seven tube genes) and can also be used for analysis. The phylogenetic relationship between different types of ST and the connection with the disease. As early as in 2003, MLST was applied to the molecular epidemiological study of Bacillus like bacteria. It has simple operation, high resolution, and more convenient for international colleagues to share data. At present, there are no related articles about the MLST of the genus of the genus of the genus, and the analysis of the epidemic of the genus in our country. The trend of the change of the situation and the Bacillus like bacteria is becoming more and more important. Therefore, we randomly selected 102 strains of clinical nose type strains spanning 11 years to carry out the MLST typing. Source and early warning provide reference.2012 Wiersinga WJ in the new England magazine reported the global trend of the global epidemic of nose like nose, estimated its incidence of 50/100000, causing the international colleagues on the study of the high level of necrosis. In China, the study of nose like necrosis has not caused enough attention to many reasons, of which the first reason is the diagnosis. In Hainan, many doctors are still unfamiliar with the noses, not to mention the medical workers in the mainland. Therefore, we are based on the database of the three largest local hospitals in Hainan, The clinical data of 170 cases of nose nose across 11 years were collected, and the risk, trend, geographical distribution, seasonal distribution and clinical characteristics of the infection in our country were summarized and analyzed, which provided reference for the clinical diagnosis and investigation and analysis of the type of nose type in China. The most difficult problem in the treatment of clinical nose type nose is the chronicity of the infection. And relapse. Even in the case of standard antibiotics, the recurrence rate is still up to 20%., and it has been reported that the Bacillus like bacteria can invade almost all the host cells, live and live in it, and cause the host cell to fuse to realize the proliferation and diffusion of the pathogen itself. This may result in the chronicity and recurrence of the infection of the nose like infection. Autophagy is one of the important ways to eliminate intracellular pathogens. Autophagosomes capture pathogens and form mature autophagic lysosomes to degrade bacteria by combining with lysosomes to resist the invasion and infection of intracellular bacteria. But some pathogens have evolved mechanisms that can resist or escape autophagy. The relationship between the autophagy of the host and the autophagy of the host and how it resists the autophagy of the host cells remains to be clarified. Our previous study has found that the type of necrosis of the nose can inhibit autophagy by inducing certain specific miRNA to inhibit the formation of autophagic and thus achieve the survival and increase in the cell. Based on the gene chip analysis of the murine macrophage RAW264.7, we found that the inhibition of autophagic digestion in mice is not by inhibiting the formation of autophagosomes, but by affecting the fusion of autophagosomes and lysosomes to inhibit the maturation of autophagosomes, and this inhibition process is also It is mediated by the specific miRNA induced by Bacillus like bacteria. This study shows that autophagy, especially complete autophagy, is very important to host cells against intracellular bacteria infection. Meanwhile, the results of previous studies also suggest that microbial induced miRNA plays an important role in affecting the host cell signaling pathway, and may be associated with a lot of microbiology. The process of infection is closely related, and it suggests that we may use the small RNA interference strategy to provide different treatment ideas for the anti microbial infection. Method 1. the retrospective analysis of Hainan type in China 1.1 was based on the database of three largest local hospitals in Hainan, and collected the clinical data of 170 cases of Nosocomia for 11 years; 1.2 All cases need clinical and laboratory confirmed diagnosis of nose like nose. 1.3 summary and analysis of the risk of infection, epidemic trend, geographical distribution, seasonal distribution, and clinical characteristics of the MLST analysis of.2. genus of genus gilli. 2.1, 102 strains of clinically isolated strains of genus nose, first performed biochemical and 16srdnapcr identification; 2.2 to ace, ghmd, gltb, LiPA, LEP A, nark, ndh seven housekeeper genes were amplified and sequenced; 2.3 on-line analysis of sequencing results, determining st type, uploading data, on-line analysis of the sequence of this study strain or MLST database sequence of synthetic genus of Bacillus genus MLST, UPGMA method constructed phylogenetic tree for traceability analysis of the collected strains, and 2.4 using MLST and 16srdn. A typing and other methods for laboratory diagnosis and traceability analysis of 1 cases of imported Genoid cases in Zhenjiang. The analysis of the 3.1 type of raw cell model and the expression spectrum chip analysis under the condition of different infection phase of the 3.1 type of the infection of the macrophage. 3.2 small RNA interference or eukaryotic expression are used to analyze the key to the autophagy signaling pathway. Protein in the cell proliferation of gangrene like; 3.3 laser confocal, high resolution microscopy and transmission electron microscopy to observe intracellular lc3b transformation, cell fusion, intracellular bacteria survival, autophagosome and autophagosysosome formation; 3.4cfu plate count is used to quantify the proliferation of intracellular gangrene, and 3.5qrt-pcr is used for quantitative mRNA level, Westernblot is used to reflect the protein level of autophagic molecules; 3.6 luciferase test is used for miR-146a target identification test; 3.7 data analysis: logsticregression for risk factors correlation analysis; e-burst for MLST typing data analysis; clcgenomicsworkbench for sequence analysis and primer design; graphpadprism5.0 statistical mapping; zen2012 Analysis confocal data; bio-radcfxmanager3.0 for quantitative PCR results analysis; adobeillustratorcs3 for picture integration; student 'st test used to detect the difference between two samples, *p0.05 has statistical significance. Results 1. the clinical retrospective study of Hainan type nose grad in China 1.1 cases of Chinese type nose is growing; 1.2 Chinese type of nose nose The bed was characterized by its own characteristics, mainly pneumonia and septicaemia, organ abscess, child infection and intracranial infection. The 1.3 type of infection was related to the immune factors of the body, a considerable part of the chronic infection; the 2.mlst analysis of 2.1102 strains of the genus acerinus was 41, and 8 new st types were found to be submitted to the database; 2.2st-4 6, st-50, st-55, st-58, ST-70 and st-1095 are the main st types of 102 strains of Bacillus like strains. Gmhd in the housekeeping gene for MLST typing showed more variation characteristics. Phylogenetic analysis found that China and Thailand were closely related to the evolution of Bacillus like strains, and may also be disseminated with other epidemic areas; 2.3 accurately identified the Jiangsu in 2014. The.3. type of the type of the type of the type of the type of the type of the type of the genus.3., the miR-146a inhibited the autophagy of the macrophage in the mouse, which is beneficial to the survival of the cell, which can induce the formation of the autophagosin of the macrophage but can coexist with it; the 3.2 autophagosomal can only be caught and restricted in the early stage of the macrophage of the mice infected with the bacillus. The survival and proliferation of the Bacillus like bacteria; the 3.3 type of the bacillus which promoted the survival of the autophagosome, but inhibited the autophagic flow; 3.4 based on the chip detection and lysosome signal transduction analysis, the inhibition of Lipa protein level may be the target of the inhibition of the autophagy of the host cell, and the former is the maturation of the autophagic lysosome. Bond protein; 3.5 at the same time, it was found that after the infection of Lipa protein, miR-146a was up-regulated with the level of Lipa protein, and 3.6 bioinformatics online analysis of Lipa might be the target protein of miR-146a and confirmed by the target gene identification experiment; the 3.7 type of the bacilli could inhibit the autophagy of mouse macrophages by the MI R-146a-Lipa signaling pathway, thus promoting it. Conclusion this study, on the one hand, enhances our understanding of the clinical knowledge of the genie like disease, understands the epidemic situation and variation characteristics of the current type of nose nose, and perfects the genetic information database of the strain of the strains of genus nose like in China, which can provide a loan for clinicians, especially the medical workers outside the epidemic area. On the other hand, the mechanism of the autophagy escape mechanism of Bacillus like bacteria, on the other hand, provides a mechanism explanation for the intractable clinical problems (the chronicity and recurrence of the infection), suggesting that the MI RNA induced by microorganism is important in the signaling pathway of the host cell. It provides a new idea for clinical treatment of M.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378

【相似文献】