本文選題：類鼻疽 + 類鼻疽菌�。� 參考：《第三軍醫(yī)大學(xué)》2016年博士論文

【摘要】：類鼻疽伯克霍爾德菌(簡(jiǎn)稱類鼻疽菌)屬兼性胞內(nèi)菌,作為人類鼻疽病的病原體,是一種環(huán)境寄生的機(jī)會(huì)致病菌,廣泛分布于東南亞和澳洲北部。我國(guó)海南、廣東和廣西等地是類鼻疽的疫源地。2016年的第1期Nature Mirobiology雜志對(duì)全球類鼻疽菌流行擴(kuò)散形勢(shì)和感染風(fēng)險(xiǎn)進(jìn)行預(yù)測(cè)分析,估算2015年在全球范圍內(nèi)類鼻疽感染病例超過16萬(wàn)人,死亡人數(shù)約9萬(wàn)人。鑒于類鼻疽的嚴(yán)重致病性,美國(guó)CDC早在2006年就將類鼻疽菌列為I類病原體嚴(yán)加防范。90年代,我國(guó)學(xué)者宋陽(yáng)等已經(jīng)報(bào)道過類鼻疽菌在海南的地理分布,當(dāng)?shù)丶部刂行囊苍M(jìn)行過類鼻疽菌的環(huán)境監(jiān)測(cè),但可查資料仍然很少,類鼻疽的擴(kuò)散之勢(shì)可能早已遠(yuǎn)超我們估計(jì)。多位點(diǎn)序列分型(MLST)是一種基于核酸序列(七個(gè)管家基因)測(cè)定的細(xì)菌分型方法,也可用于分析不同ST型之間的系統(tǒng)發(fā)育關(guān)系以及與疾病的聯(lián)系。早在2003年MLST就被應(yīng)用到類鼻疽菌的分子流行病學(xué)研究中,它操作簡(jiǎn)單,分辨力高,更便于國(guó)際同行進(jìn)行數(shù)據(jù)共享。目前,我國(guó)還沒有類鼻疽菌MLST的相關(guān)文章報(bào)道,分析我國(guó)類鼻疽的流行形勢(shì)和類鼻疽菌的變化趨勢(shì)顯得日益重要。因此,我們隨機(jī)選取了跨越11年的102株臨床類鼻疽菌株進(jìn)行了MLST分型,借此分析發(fā)現(xiàn)近年我國(guó)類鼻疽菌的變異規(guī)律和擴(kuò)散趨勢(shì),進(jìn)一步完善我國(guó)類鼻疽菌株基因信息數(shù)據(jù)庫(kù),為我國(guó)類鼻疽的溯源和預(yù)警提供參考。2012年Wiersinga WJ在新英格蘭雜志報(bào)道了類鼻疽的全球流行趨勢(shì),估算其發(fā)病率達(dá)50/100000,引起了國(guó)際同行對(duì)于類鼻疽研究的高度關(guān)注。在中國(guó),類鼻疽的研究并未引起足夠的重視有多方面的原因,其中,首要的原因是診斷困難,導(dǎo)致類鼻疽的漏診率極高。其次是類鼻疽的臨床表現(xiàn)多樣缺乏特征,易誤診為其他感染性疾病。即使在海南當(dāng)?shù)?很多醫(yī)生仍然對(duì)類鼻疽比較陌生,更不用說內(nèi)地的醫(yī)療工作者了。為此,我們基于海南當(dāng)?shù)厝畲筢t(yī)院的數(shù)據(jù)庫(kù),收集了跨越11年共計(jì)170例類鼻疽病例的臨床資料,匯總分析了我國(guó)類鼻疽感染風(fēng)險(xiǎn)、流行趨勢(shì)、地理分布、季節(jié)分布和臨床特征,為我國(guó)類鼻疽的臨床診斷和調(diào)查分析提供借鑒和參考。臨床類鼻疽治療中最難控制的問題即是感染的慢性化和復(fù)發(fā)。即使在規(guī)范抗生素用藥情況下,復(fù)發(fā)率仍達(dá)到20%。已有文獻(xiàn)報(bào)道,類鼻疽菌可以侵入幾乎所有的宿主細(xì)胞,在其中寄居和生存,并導(dǎo)致宿主細(xì)胞融合實(shí)現(xiàn)病原菌自身的增殖和擴(kuò)散。而這可能就是導(dǎo)致類鼻疽感染慢性化和易復(fù)發(fā)的重要原因。自噬是清除胞內(nèi)病原體的重要途徑之一,自噬體捕獲病原體,通過與溶酶體結(jié)合,形成成熟的自噬溶酶體降解細(xì)菌從而達(dá)到抵抗胞內(nèi)菌入侵和感染的目的。但是有些病原體已經(jīng)進(jìn)化出能夠抵抗或者逃避自噬清除的機(jī)制,而類鼻疽與宿主自噬之間的關(guān)系以及它是如何抵制宿主細(xì)胞自噬清除的機(jī)制還有待進(jìn)一步闡明。我們前期的研究已經(jīng)發(fā)現(xiàn)類鼻疽可以通過誘導(dǎo)某些特殊mirna來(lái)抑制人肺上皮細(xì)胞的自噬水平,抑制自噬體的形成從而實(shí)現(xiàn)在胞內(nèi)的生存和增殖�；趯�(duì)類鼻疽菌感染的小鼠巨噬細(xì)胞raw264.7的基因芯片分析,我們發(fā)現(xiàn),類鼻疽菌抑制小鼠細(xì)胞的自噬消化并不是通過抑制自噬體形成,而是通過影響自噬體與溶酶體的融合、抑制自噬溶酶體的成熟來(lái)實(shí)現(xiàn)的,而且這一抑制過程也是由類鼻疽菌誘導(dǎo)的特異性mirna所介導(dǎo)。這項(xiàng)研究表明,自噬,尤其是完整的自噬對(duì)于宿主細(xì)胞對(duì)抗胞內(nèi)菌感染是非常重要的。同時(shí),前期研究結(jié)果也提示我們,微生物誘導(dǎo)的mirna在影響宿主細(xì)胞信號(hào)通路中具有重要作用,可能與許多的微生物感染進(jìn)程緊密相關(guān),也暗示我們利用小rna干擾策略或許能夠給對(duì)抗微生物感染提供不一樣的治療思路。方法1.中國(guó)海南類鼻疽-回顧性分析1.1基于海南當(dāng)?shù)厝畲筢t(yī)院的數(shù)據(jù)庫(kù),收集跨越11年共計(jì)170例類鼻疽病例的臨床資料;1.2所有病例均需臨床和實(shí)驗(yàn)室確認(rèn)類鼻疽診斷;1.3匯總分析我國(guó)類鼻疽感染風(fēng)險(xiǎn)、流行趨勢(shì)、地理分布、季節(jié)分布和臨床特征。2.類鼻疽菌的mlst分析2.1對(duì)102株臨床分離的類鼻疽菌株首先進(jìn)行生化與16srdnapcr鑒定;2.2對(duì)類鼻疽菌ace,ghmd,gltb,lipa,lepa,nark,ndh七個(gè)管家基因進(jìn)行擴(kuò)增和測(cè)序分析;2.3在線分析測(cè)序結(jié)果,確定st型,上傳數(shù)據(jù),在線對(duì)本次研究菌株序列或者綜合類鼻疽菌mlst數(shù)據(jù)庫(kù)序列進(jìn)行e-burst分析,upgma方法構(gòu)建系統(tǒng)進(jìn)化樹對(duì)收集的菌株進(jìn)行溯源分析等;2.4運(yùn)用mlst及16srdna分型等方法對(duì)鎮(zhèn)江1例輸入性類鼻疽病例進(jìn)行實(shí)驗(yàn)室確診和溯源分析。3.類鼻疽菌抑制巨噬細(xì)胞自噬研究3.1類鼻疽菌感染raw細(xì)胞模型和不同感染時(shí)相條件下的表達(dá)譜芯片分析;3.2小rna干擾或者真核過表達(dá),用于分析自噬信號(hào)通路關(guān)鍵蛋白在類鼻疽的胞內(nèi)增殖作用;3.3激光共聚焦、高分辨顯微鏡和透射電鏡用于觀察胞內(nèi)lc3b轉(zhuǎn)化,細(xì)胞融合,胞內(nèi)菌生存情況,自噬體以及自噬溶酶體的形成;3.4cfu平板計(jì)數(shù)用于定量細(xì)胞內(nèi)類鼻疽菌的增殖情況;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熒光素酶實(shí)驗(yàn)用于mir-146a靶基因鑒定試驗(yàn);3.7數(shù)據(jù)分析:logsticregression用于危險(xiǎn)因素相關(guān)性分析;e-burst用于mlst分型數(shù)據(jù)分析;clcgenomicsworkbench用于序列分析和引物設(shè)計(jì);graphpadprism5.0統(tǒng)計(jì)作圖;zen2012分析共聚焦數(shù)據(jù);bio-radcfxmanager3.0用于定量pcr結(jié)果分析;adobeillustratorcs3進(jìn)行圖片整合;student’st檢驗(yàn)用于檢測(cè)兩樣本之間差異,*p0.05有統(tǒng)計(jì)學(xué)意義。結(jié)果1.中國(guó)海南類鼻疽臨床回顧性研究1.1中國(guó)類鼻疽病例呈現(xiàn)增長(zhǎng)態(tài)勢(shì);1.2中國(guó)類鼻疽臨床表現(xiàn)有自身特點(diǎn),主要以肺炎和敗血癥為主,臟器膿腫、兒童感染和顱內(nèi)感染較少見;1.3類鼻疽的感染與機(jī)體免疫因素相關(guān),相當(dāng)一部分呈現(xiàn)慢性感染;2.mlst分析2.1102株類鼻疽菌st型別多達(dá)41種,發(fā)現(xiàn)8種新的st型,均已提交至數(shù)據(jù)庫(kù);2.2st-46,st-50,st-55,st-58,st-70和st-1095是102株類鼻疽菌中的主要st型,用于mlst分型的管家基因中g(shù)mhd表現(xiàn)出更多的變異特征,系統(tǒng)發(fā)生分析發(fā)現(xiàn),中國(guó)與泰國(guó)在類鼻疽菌的進(jìn)化上關(guān)系緊密,與其它疫區(qū)也可能存在交流播散;2.3準(zhǔn)確鑒定了2014年江蘇輸入性類鼻疽病例并成功指導(dǎo)其處置。3.類鼻疽菌誘導(dǎo)的mir-146a抑制小鼠巨噬細(xì)胞自噬有利于其胞內(nèi)的生存3.1類鼻疽菌可以誘導(dǎo)巨噬細(xì)胞自噬體的形成但是卻能與之共處;3.2自噬體只能在類鼻疽菌感染小鼠巨噬細(xì)胞初期捕獲和限制類鼻疽菌的生存和增殖;3.3類鼻疽菌雖然促進(jìn)了自噬體的生存,但是卻抑制了自噬流;3.4基于芯片檢測(cè)和溶酶體信號(hào)通路表達(dá)譜分析,Lipa蛋白水平受抑可能是類鼻疽菌抑制宿主細(xì)胞自噬的效應(yīng)靶點(diǎn),而前者是細(xì)胞自噬溶酶體成熟的關(guān)鍵蛋白;3.5同時(shí)發(fā)現(xiàn),類鼻疽菌感染后,伴隨著Lipa蛋白水平受抑,miR-146a呈現(xiàn)上調(diào);3.6生物信息學(xué)在線分析Lipa可能是miR-146a的靶蛋白,并經(jīng)靶基因鑒定實(shí)驗(yàn)證實(shí);3.7類鼻疽菌可借助mi R-146a-Lipa信號(hào)通路抑制小鼠巨噬細(xì)胞自噬,從而促進(jìn)其在宿主細(xì)胞中的生存和增殖。結(jié)論本研究,一方面增強(qiáng)了我們對(duì)于類鼻疽的臨床認(rèn)識(shí),了解了當(dāng)前類鼻疽的流行形勢(shì)和變異特點(diǎn),完善了我國(guó)類鼻疽菌株基因信息數(shù)據(jù)庫(kù),可為臨床醫(yī)生尤其是疫區(qū)以外的醫(yī)務(wù)工作者對(duì)類鼻疽的診斷提供借鑒和參考,同時(shí)為我國(guó)類鼻疽的溯源和預(yù)警提供指導(dǎo);另一方面,通過深入探討類鼻疽菌的自噬逃逸機(jī)制,對(duì)類鼻疽的臨床棘手問題(感染慢性化和復(fù)發(fā))提供了機(jī)理解釋,提示我們微生物誘導(dǎo)的mi RNA在影響宿主細(xì)胞信號(hào)通路中的重要作用,為臨床對(duì)抗類鼻疽菌感染治療提供了新的思路。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R378

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