本文選題：類鼻疽桿菌　切入點(diǎn)：自噬　出處：《第三軍醫(yī)大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：類鼻疽(Melioidosis)是由類鼻疽桿菌(Burkholderia pseudomallei,全稱為類鼻疽伯克霍爾德氏菌)所引起的一種人獸共患傳染病。該疾病可通過呼吸道、消化道、經(jīng)皮傳播,主要侵犯人體肺部,可造成肺炎、肺膿腫以及肺部空洞,還可引起皮膚、肝臟、脾臟等多器官膿腫,嚴(yán)重者可快速發(fā)展為敗血癥,由于其診斷難、致病強(qiáng)、治療難、潛伏長,死亡率高達(dá)40%;其流行區(qū)域主要集中分布在南、北緯二十度內(nèi),涵蓋澳大利亞、新加坡、越南、柬埔寨、老撾、馬來西亞、泰國東北部等地區(qū)以及我國海南、香港、臺灣、廣東等南海及臺海等主要戰(zhàn)略方向,現(xiàn)越來越多的證據(jù)表明它是一種正在擴(kuò)散的人獸共患傳染病。海南國際旅游島的開發(fā)、“一帶一路”的建設(shè)以及南海海洋主權(quán)維權(quán)和經(jīng)濟(jì)安全保障等已上升至國家戰(zhàn)略要求,加強(qiáng)類鼻疽研究具有重要的社會意義和軍事價(jià)值。深入研究B.pseudomallei感染致病機(jī)理、發(fā)展新的有效的B.pseudomallei感染治療手段迫在眉睫。B.pseudomallei作為胞內(nèi)感染病原菌,有效逃逸機(jī)體免疫清除是B.pseudomallei感染與致病的前提。天然免疫作為人體抵御病原微生物侵襲的第一道防線,在抗感染中發(fā)揮著至關(guān)重要的作用。自噬作為天然免疫中重要的組成部分,同時(shí)也作為機(jī)體自我保護(hù)的生理學(xué)行為,在抗病原微生物感染以及其導(dǎo)致的炎癥中發(fā)揮關(guān)鍵作用。在類鼻疽桿菌的自噬研究中,早期發(fā)現(xiàn)B.pseudomallei感染可誘導(dǎo)小鼠巨噬細(xì)胞RAW264.7產(chǎn)生自噬,但隨著感染的持續(xù),自噬出現(xiàn)抑制,RAW264.7細(xì)胞對B.pseudomallei清除能力減弱。課題組前期研究也發(fā)現(xiàn),B.pseudomallei感染肺上皮細(xì)胞A549后,自噬水平較未感染組明顯減弱;通過m RNA表達(dá)譜芯片篩選發(fā)現(xiàn)自噬相關(guān)基因ATG10下調(diào)最顯著。那么,B.pseudomallei是如何調(diào)控及抑制自噬,以利于其在宿主細(xì)胞中的存活和復(fù)制,使得機(jī)體不能有效清除B.pseudomallei,從而導(dǎo)致持續(xù)性感染的呢?mi RNAs作為病原與宿主相互作用的信號調(diào)節(jié)分子,可通過介導(dǎo)清除病原體的自噬反應(yīng),發(fā)揮抗病原微生物的感染免疫調(diào)節(jié)作用。為了探究B.pseudomallei感染過程中調(diào)控自噬的具體機(jī)制,以及mi RNAs是否參與、如何參與自噬調(diào)節(jié)的分子機(jī)制,我們以肺上皮細(xì)胞A549為細(xì)胞模型,開展了B.pseudomallei感染A549細(xì)胞后mi RNA表達(dá)譜芯片的篩選,對上調(diào)的mi RNA進(jìn)行生物信息學(xué)分析,結(jié)果發(fā)現(xiàn)三種未被報(bào)道的mi RNAs:MIR4458、MIR4667-5p、MIR4668-5p,它們可在3個(gè)不同的感染時(shí)間點(diǎn)分別靶向自噬信號通路的關(guān)鍵分子ATG10,提示我們B.pseudomallei可能通過改變宿主細(xì)胞的mi RNAs表達(dá)譜,干擾宿主細(xì)胞自噬,實(shí)現(xiàn)逃避機(jī)體免疫清除的目的。因此,明確這些mi RNAs介導(dǎo)的自噬抑制在類鼻疽桿菌感染免疫逃逸中的機(jī)制,將有助于開發(fā)作用于mi RNAs或其靶標(biāo)的新的治療手段,尤其是針對類鼻疽桿菌此種耐藥性強(qiáng)、胞內(nèi)感染的病原,臨床意義更為重要。【研究目的】1.明確B.pseudomallei感染A549細(xì)胞后自噬受抑的生物學(xué)現(xiàn)象;2.明確B.pseudomallei感染A549細(xì)胞后自噬相關(guān)蛋白ATG10的表達(dá)變化;3.探討MIR4458、MIR4667-5p、MIR4668-5p負(fù)性調(diào)控自噬的具體機(jī)制以及對細(xì)胞清除胞內(nèi)B.pseudomallei的影響;4.探討B(tài).pseudomallei感染對MIR4458、MIR4667-5p、MIR4668-5p轉(zhuǎn)錄調(diào)控的分子機(jī)制。【研究方法】1.B.pseudomallei感染A549細(xì)胞后對細(xì)胞自噬的影響首先建立B.pseudomallei感染A549細(xì)胞模型,通過透射電鏡、Western blot、激光共聚焦檢測B.pseudomallei感染A549細(xì)胞后細(xì)胞自噬水平變化情況;再者,藥物激活或抑制自噬的條件下,通過細(xì)菌胞內(nèi)計(jì)數(shù)實(shí)驗(yàn)評價(jià)自噬對A549細(xì)胞清除胞內(nèi)B.pseudomallei能力的影響。2.B.pseudomallei感染A549細(xì)胞自噬相關(guān)基因ATG10下調(diào)特點(diǎn)研究通過m RNA表達(dá)譜芯片篩選,發(fā)現(xiàn)自噬相關(guān)蛋白ATG10下調(diào)最明顯;結(jié)合表達(dá)譜芯片篩選結(jié)果,在m RNA和蛋白水平分析ATG10的時(shí)空表達(dá)特點(diǎn);同時(shí),構(gòu)建ATG10真核表達(dá)質(zhì)粒,回復(fù)下調(diào)的ATG10后,觀察并檢測自噬和細(xì)菌清除的變化。3.MIR4458、MIR4667-5p和MIR4668-5p通過ATG10介導(dǎo)抑制自噬的機(jī)制研究結(jié)合mi RNA表達(dá)譜芯片篩選和生物信息學(xué)預(yù)測分析,發(fā)現(xiàn)MIR4458、MIR4667-5p和MIR4668-5p可靶向自噬相關(guān)基因ATG10;首先構(gòu)建熒光素酶報(bào)告載體進(jìn)一步鑒定ATG10是MIR4458、MIR4667-5p和MIR4668-5p的靶基因;分別過表達(dá)或抑制MIR4458、MIR4667-5p和MIR4668-5p,通過Western blot、激光共聚焦以及胞內(nèi)細(xì)菌計(jì)數(shù)實(shí)驗(yàn)觀察mi RNAs對自噬和細(xì)菌清除的影響。4.B.pseudomallei感染A549細(xì)胞后上調(diào)MIR4458、MIR4667-5p、MIR4668-5p表達(dá)的轉(zhuǎn)錄調(diào)控分子機(jī)制研究利用UCSC生物信息學(xué)軟件分析和預(yù)測MIR4458、MIR4667-5p、MIR4668-5p啟動子區(qū)域的轉(zhuǎn)錄調(diào)控元件,發(fā)現(xiàn)均含有較大的Cp G島;首先通過BSP和DNMT活性檢測,觀察B.pseudomallei感染后MIR4458、MIR4667-5p和MIR4668-5p的Cp G島甲基化水平改變;再者,利用去甲基化藥物5-Aza-Cd R處理,通過MSP和q RT-PCR實(shí)驗(yàn),檢測降低甲基化水平對MIR4458、MIR4667-5p和MIR4668-5p表達(dá)的影響�！狙芯拷Y(jié)果】1.B.pseudomallei感染A549細(xì)胞后自噬受抑通過透射電鏡、Western blot、激光共聚焦實(shí)驗(yàn),發(fā)現(xiàn)B.pseudomallei感染A549細(xì)胞后自噬受抑;另外,在誘導(dǎo)或抑制自噬的條件下,通過胞內(nèi)細(xì)菌計(jì)數(shù)實(shí)驗(yàn)發(fā)現(xiàn)激活自噬可以增強(qiáng)宿主細(xì)胞對B.pseudomallei的胞內(nèi)清除能力。2.B.pseudomallei通過下調(diào)ATG10抑制自噬通過m RNA表達(dá)譜芯片篩選,發(fā)現(xiàn)B.pseudomallei感染后自噬相關(guān)蛋白ATG10下調(diào)最明顯;利用q RT-PCR和Western blot實(shí)驗(yàn)分析,ATG10在m RNA和蛋白水平上均發(fā)生明顯下調(diào);同時(shí),過表達(dá)ATG10可增強(qiáng)細(xì)胞自噬以及對B.pseudomallei的胞內(nèi)清除能力。3.B.pseudomallei通過上調(diào)MIR4458、MIR4667-5p、MIR4668-5p抑制自噬通過生物信息學(xué)分析以及雙熒光素酶報(bào)告實(shí)驗(yàn)、Western blot及q RT-PCR實(shí)驗(yàn),預(yù)測并鑒定了ATG10是MIR4458、MIR4667-5p、MIR4668-5p的靶基因;分別過表達(dá)或抑制這3個(gè)mi RNA后,發(fā)現(xiàn)MIR4458、MIR4667-5p和MIR4668-5p通過ATG10介導(dǎo)了抑制宿主細(xì)胞自噬以及對B.pseudomallei胞內(nèi)清除能力。4.B.pseudomallei感染后引起的MIR4458、MIR4667-5p、MIR4668-5p啟動子區(qū)域Cp G島甲基化水平降低與其表達(dá)調(diào)控有關(guān)通過BSP和DNMT活性檢測,發(fā)現(xiàn)B.pseudomallei感染后MIR4458、MIR4667-5p和MIR4668-5p的Cp G島甲基化水平降低;同時(shí),去甲基化藥物5-Aza-Cd R處理后,MSP分析MIR4458、MIR4667-5p和MIR4668-5p的Cp G島發(fā)生去甲基化,進(jìn)一步q RT-PCR檢測發(fā)現(xiàn)MIR4458、MIR4667-5p和MIR4668-5p的表達(dá)均增加�！局饕Y(jié)論】B.pseudomallei感染可通過甲基化調(diào)控方式,誘導(dǎo)MIR4458、MIR4667-5p和MIR4668-5p的上調(diào),進(jìn)而實(shí)時(shí)、連續(xù)的抑制其靶基因ATG10的表達(dá),負(fù)性調(diào)控自噬,從而影響宿主細(xì)胞對B.pseudomallei的清除,促進(jìn)其胞內(nèi)存活。【研究意義】本文的研究展示了一個(gè)新的調(diào)控自噬的機(jī)制,其創(chuàng)新性主要在于探討了“B.pseudomallei、mi RNA與自噬”的作用模式,以mi RNA為切入點(diǎn),初步闡明了B.pseudomallei干擾宿主細(xì)胞自噬,實(shí)現(xiàn)逃避宿主免疫清除的分子機(jī)制。該研究為類鼻疽桿菌感染的治療提供了一個(gè)新的治療靶標(biāo),同時(shí)也為其他胞內(nèi)病原感染的治療提供了借鑒。
[Abstract]:Melioidosis (Melioidosis) by Burkholderia pseudomallei (Burkholderia pseudomallei, Burke Holder's name for melioidosis bacteria) is a zoonotic infectious disease caused by the disease. Through the respiratory tract, digestive tract, percutaneous spread, mainly involving the lungs, can cause pneumonia, lung abscess and pulmonary cavity, can also cause the skin, liver, spleen and other organs abscess, severe cases can be rapid development of sepsis, due to the difficult diagnosis, pathogenesis, treatment is difficult, long latency, high mortality rate of 40%; the epidemic area mainly concentrated in the south, twenty degrees north latitude, including Australia, Singapore, Vietnam, Kampuchea, Laos, Malaysia. Northeast Thailand and other regions as well as China's Hainan, Hongkong, Taiwan, Guangdong and other South China Sea and Taiwan and other major strategic direction, now more and more evidence that it is a zoonotic infectious disease spread in Hainan international travel. The development of the island, "the construction of The Belt and Road" and the South China Sea sovereignty rights and economic security has risen to national strategic requirements, has important social significance and value to strengthen the military research of melioidosis. An in-depth study of the pathogenic mechanism of B.pseudomallei infection, the development of new and effective treatment for B.pseudomallei infection of.B.pseudomallei infection pathogens as imminent intracellular, effective escape immune clearance is the premise of B.pseudomallei infection and pathogenicity. The natural immune as the body's first line of defense against the invasion of pathogenic microorganisms, plays an important role in anti infection. Autophagy as an important component of innate immunity, but also as a physiological organism self protective behavior, play a key role in anti microbial infection and inflammation of the result. In the autophagy of melioidosis in early That B.pseudomallei infection can induce RAW264.7 mice macrophage autophagy, but with persistent infection, autophagy appears to inhibit RAW264.7 cells on B.pseudomallei, scavenging ability weakened. Previous studies also found that B.pseudomallei infection of lung epithelial cells after A549, autophagy level is not infected group was significantly decreased; microarray screening found that autophagy related gene ATG10 by the expression of RNA m significantly. So, B.pseudomallei is how to control and inhibition of autophagy, which is beneficial to the survival in the host cell and replicate, leaves the body unable to effectively remove B.pseudomallei, resulting in persistent infection? Signal mi RNAs as the pathogen host interactions of regulatory molecules, autophagy can remove pathogens through the guide dielectric, infection and immunity against pathogenic microorganisms play a regulatory role. In order to explore the B.pseudomallei infection process control The specific mechanisms of autophagy, and MI RNAs is involved, how to participate in the molecular mechanism of autophagy in lung epithelial cells, we A549 cell model, carried out microarray expression screening mi RNA B.pseudomallei in A549 cells infected with MI RNA on the upregulation of the bioinformatics analysis results showed that three kinds of MI has not been reported RNAs:MIR4458, MIR4667-5p, MIR4668-5p, which is available in 3 different time points of infection of ATG10 targeting key molecules in autophagy pathway respectively, indicating that B.pseudomallei may alter the host cell mi RNAs expression, interfering host cell autophagy, achieve the purpose of evading the immune clearance. Therefore, these clear mi RNAs mediated autophagy the mechanism of inhibition of Burkholderia pseudomallei infection in immune escape, there will be a new treatment to help in the development of MI RNAs or its target, especially for melioidosis This resistance is strong, intracellular pathogen infection, the clinical significance is more important. [Objective] to study biological phenomenon of 1. clear B.pseudomallei infected A549 cells autophagy inhibition; expression of autophagy related protein ATG10 2. clear B.pseudomallei infected A549 cells; 3. of MIR4458, MIR4667-5p, MIR4668-5p specific mechanisms of negative regulation of autophagy and effect on cell clearance of intracellular B.pseudomallei; 4. to explore B.pseudomallei infection of MIR4458, MIR4667-5p, molecular mechanism of the transcriptional regulation of MIR4668-5p. [Methods] 1.B.pseudomallei infection of A549 cells after effects on autophagy first establish A549 cell model of B.pseudomallei infection by transmission electron microscopy, Western blot, confocal laser changes of autophagy in detection of B.pseudomallei infection A549 cells; moreover, drug activation or inhibition of autophagy conditions by bacteria Cell counting experiment evaluation of autophagy in influencing intracellular B.pseudomallei scavenging ability of A549 cells in.2.B.pseudomallei infected A549 cell autophagy related gene ATG10 by screening characteristics of microarray by M RNA found that expression of autophagy related protein ATG10 decreased most obviously; combined with microarray results, ATG10 analysis of the spatiotemporal expression characteristics in M RNA and protein levels; at the same time, the construction of eukaryotic expression plasmid of ATG10, down-regulation of ATG10 after recovery, observe and detect autophagy and bacterial clearance changes of.3.MIR4458, MIR4667-5p and MIR4668-5p through ATG10 mediated inhibition mechanism of macrophages with MI from RNA microarray and bioinformatics prediction and analysis, found that MIR4458, MIR4667-5p and MIR4668-5p can target the autophagy related ATG10 gene; luciferase reporter vector constructed further identification of ATG10 is MIR4458, the target gene MIR4667-5p and MIR4668-5p; Respectively over expression or inhibition of MIR4458, MIR4667-5p and MIR4668-5p, by Western blot, confocal laser and intracellular bacterial count experimental observations of MI RNAs on autophagy and the effect of bacterial clearance of.4.B.pseudomallei infected A549 cells by upregulation of MIR4458, MIR4667-5p, MIR4668-5p expression of the molecular mechanism of transcriptional regulation by UCSC bioinformatics software analysis and prediction of MIR4458, MIR4667-5p MIR4668-5p, start the transcription regulatory element sub region, was found to contain larger Cp G island; first detected by BSP and DNMT activity, B.pseudomallei was observed after MIR4458 infection, Cp G island methylation levels of MIR4667-5p and MIR4668-5p change; moreover, the use of demethylation drugs 5-Aza-Cd R, by MSP and Q RT-PCR experiment, detection reduce the methylation level of MIR4458, the expression of MIR4667-5p and MIR4668-5p. [results] 1.B.pseudomallei infection of A549 cells After the inhibition of autophagy by transmission electron microscopy, Western blot, confocal laser experiment, found that B.pseudomallei infected A549 cells after inhibition of autophagy; in addition, in the induction or inhibition of autophagy conditions through intracellular bacterial counting experiments found that activation of autophagy can enhance host cells of B.pseudomallei cells through down-regulation of ATG10.2.B.pseudomallei scavenging ability of inhibiting autophagy screening the expression of RNA by M microarray, found that B.pseudomallei infection of autophagy related protein ATG10 decreased most obviously; analysis by Q RT-PCR and Western blot experiments, ATG10 occurred in M RNA and protein level were significantly reduced; at the same time, overexpression of ATG10 can enhance autophagy and B.pseudomallei on intracellular scavenging capacity by increasing the expression of.3.B.pseudomallei MIR4458 MIR4667-5p MIR4668-5p, inhibition of autophagy by bioinformatics analysis and dual luciferase reporter assay, Western and blot Q RT-PCR experiment, prediction and identification of ATG10 is MIR4458, MIR4667-5p, MIR4668-5p target genes respectively; overexpression or inhibition of the 3 mi RNA, MIR4667-5p MIR4668-5p and MIR4458 was found by ATG10 mediated inhibition of autophagy in host cells and intracellular B.pseudomallei removal can cause stress after.4.B.pseudomallei infection, MIR4458, MIR4667-5p, MIR4668-5p start reducing the promoter region of Cp G island methylation and its expression regulation by detecting BSP and DNMT activity, found after the infection of B.pseudomallei MIR4458, Cp G island methylation level of MIR4667-5p and MIR4668-5p decreased; meanwhile, demethylation drugs 5-Aza-Cd after R treatment, MSP MIR4458 analysis, MIR4667-5p and MIR4668-5p Cp G to the island further Q methylation, RT-PCR detected MIR4458 expression of MIR4667-5p and MIR4668-5p were increased. [Conclusion] B.pseudomallei infection mainly through methylation 璋冩帶鏂瑰紡,璇卞MIR4458,MIR4667-5p鍜孧IR4668-5p鐨勪笂璋，

本文編號：1632369