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

【摘要】：類鼻疽伯克霍爾德菌(簡稱類鼻疽菌)屬兼性胞內菌,作為人類鼻疽病的病原體,是一種環(huán)境寄生的機會致病菌,廣泛分布于東南亞和澳洲北部。我國海南、廣東和廣西等地是類鼻疽的疫源地。2016年的第1期Nature Mirobiology雜志對全球類鼻疽菌流行擴散形勢和感染風險進行預測分析,估算2015年在全球范圍內類鼻疽感染病例超過16萬人,死亡人數(shù)約9萬人。鑒于類鼻疽的嚴重致病性,美國CDC早在2006年就將類鼻疽菌列為I類病原體嚴加防范。90年代,我國學者宋陽等已經(jīng)報道過類鼻疽菌在海南的地理分布,當?shù)丶部刂行囊苍M行過類鼻疽菌的環(huán)境監(jiān)測,但可查資料仍然很少,類鼻疽的擴散之勢可能早已遠超我們估計。多位點序列分型(MLST)是一種基于核酸序列(七個管家基因)測定的細菌分型方法,也可用于分析不同ST型之間的系統(tǒng)發(fā)育關系以及與疾病的聯(lián)系。早在2003年MLST就被應用到類鼻疽菌的分子流行病學研究中,它操作簡單,分辨力高,更便于國際同行進行數(shù)據(jù)共享。目前,我國還沒有類鼻疽菌MLST的相關文章報道,分析我國類鼻疽的流行形勢和類鼻疽菌的變化趨勢顯得日益重要。因此,我們隨機選取了跨越11年的102株臨床類鼻疽菌株進行了MLST分型,借此分析發(fā)現(xiàn)近年我國類鼻疽菌的變異規(guī)律和擴散趨勢,進一步完善我國類鼻疽菌株基因信息數(shù)據(jù)庫,為我國類鼻疽的溯源和預警提供參考。2012年Wiersinga WJ在新英格蘭雜志報道了類鼻疽的全球流行趨勢,估算其發(fā)病率達50/100000,引起了國際同行對于類鼻疽研究的高度關注。在中國,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?shù)?很多醫(yī)生仍然對類鼻疽比較陌生,更不用說內地的醫(yī)療工作者了。為此,我們基于海南當?shù)厝畲筢t(yī)院的數(shù)據(jù)庫,收集了跨越11年共計170例類鼻疽病例的臨床資料,匯總分析了我國類鼻疽感染風險、流行趨勢、地理分布、季節(jié)分布和臨床特征,為我國類鼻疽的臨床診斷和調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發(fā)。即使在規(guī)范抗生素用藥情況下,復發(fā)率仍達到20%。已有文獻報道,類鼻疽菌可以侵入幾乎所有的宿主細胞,在其中寄居和生存,并導致宿主細胞融合實現(xiàn)病原菌自身的增殖和擴散。而這可能就是導致類鼻疽感染慢性化和易復發(fā)的重要原因。自噬是清除胞內病原體的重要途徑之一,自噬體捕獲病原體,通過與溶酶體結合,形成成熟的自噬溶酶體降解細菌從而達到抵抗胞內菌入侵和感染的目的。但是有些病原體已經(jīng)進化出能夠抵抗或者逃避自噬清除的機制,而類鼻疽與宿主自噬之間的關系以及它是如何抵制宿主細胞自噬清除的機制還有待進一步闡明。我們前期的研究已經(jīng)發(fā)現(xiàn)類鼻疽可以通過誘導某些特殊mirna來抑制人肺上皮細胞的自噬水平,抑制自噬體的形成從而實現(xiàn)在胞內的生存和增殖。基于對類鼻疽菌感染的小鼠巨噬細胞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實現(xiàn)的,而且這一抑制過程也是由類鼻疽菌誘導的特異性mirna所介導。這項研究表明,自噬,尤其是完整的自噬對于宿主細胞對抗胞內菌感染是非常重要的。同時,前期研究結果也提示我們,微生物誘導的mirna在影響宿主細胞信號通路中具有重要作用,可能與許多的微生物感染進程緊密相關,也暗示我們利用小rna干擾策略或許能夠給對抗微生物感染提供不一樣的治療思路。方法1.中國海南類鼻疽-回顧性分析1.1基于海南當?shù)厝畲筢t(yī)院的數(shù)據(jù)庫,收集跨越11年共計170例類鼻疽病例的臨床資料;1.2所有病例均需臨床和實驗室確認類鼻疽診斷;1.3匯總分析我國類鼻疽感染風險、流行趨勢、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102株臨床分離的類鼻疽菌株首先進行生化與16srdnapcr鑒定;2.2對類鼻疽菌ace,ghmd,gltb,lipa,lepa,nark,ndh七個管家基因進行擴增和測序分析;2.3在線分析測序結果,確定st型,上傳數(shù)據(jù),在線對本次研究菌株序列或者綜合類鼻疽菌mlst數(shù)據(jù)庫序列進行e-burst分析,upgma方法構建系統(tǒng)進化樹對收集的菌株進行溯源分析等;2.4運用mlst及16srdna分型等方法對鎮(zhèn)江1例輸入性類鼻疽病例進行實驗室確診和溯源分析。3.類鼻疽菌抑制巨噬細胞自噬研究3.1類鼻疽菌感染raw細胞模型和不同感染時相條件下的表達譜芯片分析;3.2小rna干擾或者真核過表達,用于分析自噬信號通路關鍵蛋白在類鼻疽的胞內增殖作用;3.3激光共聚焦、高分辨顯微鏡和透射電鏡用于觀察胞內lc3b轉化,細胞融合,胞內菌生存情況,自噬體以及自噬溶酶體的形成;3.4cfu平板計數(shù)用于定量細胞內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熒光素酶實驗用于mir-146a靶基因鑒定試驗;3.7數(shù)據(jù)分析:logsticregression用于危險因素相關性分析;e-burst用于mlst分型數(shù)據(jù)分析;clcgenomicsworkbench用于序列分析和引物設計;graphpadprism5.0統(tǒng)計作圖;zen2012分析共聚焦數(shù)據(jù);bio-radcfxmanager3.0用于定量pcr結果分析;adobeillustratorcs3進行圖片整合;student’st檢驗用于檢測兩樣本之間差異,*p0.05有統(tǒng)計學意義。結果1.中國海南類鼻疽臨床回顧性研究1.1中國類鼻疽病例呈現(xiàn)增長態(tài)勢;1.2中國類鼻疽臨床表現(xiàn)有自身特點,主要以肺炎和敗血癥為主,臟器膿腫、兒童感染和顱內感染較少見;1.3類鼻疽的感染與機體免疫因素相關,相當一部分呈現(xiàn)慢性感染;2.mlst分析2.1102株類鼻疽菌st型別多達41種,發(fā)現(xiàn)8種新的st型,均已提交至數(shù)據(jù)庫;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gmhd表現(xiàn)出更多的變異特征,系統(tǒng)發(fā)生分析發(fā)現(xiàn),中國與泰國在類鼻疽菌的進化上關系緊密,與其它疫區(qū)也可能存在交流播散;2.3準確鑒定了2014年江蘇輸入性類鼻疽病例并成功指導其處置。3.類鼻疽菌誘導的mir-146a抑制小鼠巨噬細胞自噬有利于其胞內的生存3.1類鼻疽菌可以誘導巨噬細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類鼻疽菌雖然促進了自噬體的生存,但是卻抑制了自噬流;3.4基于芯片檢測和溶酶體信號通路表達譜分析,Lipa蛋白水平受抑可能是類鼻疽菌抑制宿主細胞自噬的效應靶點,而前者是細胞自噬溶酶體成熟的關鍵蛋白;3.5同時發(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;3.6生物信息學在線分析Lipa可能是miR-146a的靶蛋白,并經(jīng)靶基因鑒定實驗證實;3.7類鼻疽菌可借助mi R-146a-Lipa信號通路抑制小鼠巨噬細胞自噬,從而促進其在宿主細胞中的生存和增殖。結論本研究,一方面增強了我們對于類鼻疽的臨床認識,了解了當前類鼻疽的流行形勢和變異特點,完善了我國類鼻疽菌株基因信息數(shù)據(jù)庫,可為臨床醫(yī)生尤其是疫區(qū)以外的醫(yī)務工作者對類鼻疽的診斷提供借鑒和參考,同時為我國類鼻疽的溯源和預警提供指導;另一方面,通過深入探討類鼻疽菌的自噬逃逸機制,對類鼻疽的臨床棘手問題(感染慢性化和復發(fā))提供了機理解釋,提示我們微生物誘導的mi RNA在影響宿主細胞信號通路中的重要作用,為臨床對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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R378

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