miRNAs介导的自噬抑制在类鼻疽杆菌感染免疫逃逸中的作用机制研究

发布时间：2018-03-19 02:15

  本文选题：类鼻疽杆菌　切入点：自噬　出处：《第三军医大学》2015年博士论文　论文类型：学位论文

【摘要】：类鼻疽(Melioidosis)是由类鼻疽杆菌(Burkholderia pseudomallei,全称为类鼻疽伯克霍尔德氏菌)所引起的一种人兽共患传染病。该疾病可通过呼吸道、消化道、经皮传播,主要侵犯人体肺部,可造成肺炎、肺脓肿以及肺部空洞,还可引起皮肤、肝脏、脾脏等多器官脓肿,严重者可快速发展为败血症,由于其诊断难、致病强、治疗难、潜伏长,死亡率高达40%;其流行区域主要集中分布在南、北纬二十度内,涵盖澳大利亚、新加坡、越南、柬埔寨、老挝、马来西亚、泰国东北部等地区以及我国海南、香港、台湾、广东等南海及台海等主要战略方向,现越来越多的证据表明它是一种正在扩散的人兽共患传染病。海南国际旅游岛的开发、“一带一路”的建设以及南海海洋主权维权和经济安全保障等已上升至国家战略要求,加强类鼻疽研究具有重要的社会意义和军事价值。深入研究B.pseudomallei感染致病机理、发展新的有效的B.pseudomallei感染治疗手段迫在眉睫。B.pseudomallei作为胞内感染病原菌,有效逃逸机体免疫清除是B.pseudomallei感染与致病的前提。天然免疫作为人体抵御病原微生物侵袭的第一道防线,在抗感染中发挥着至关重要的作用。自噬作为天然免疫中重要的组成部分,同时也作为机体自我保护的生理学行为,在抗病原微生物感染以及其导致的炎症中发挥关键作用。在类鼻疽杆菌的自噬研究中,早期发现B.pseudomallei感染可诱导小鼠巨噬细胞RAW264.7产生自噬,但随着感染的持续,自噬出现抑制,RAW264.7细胞对B.pseudomallei清除能力减弱。课题组前期研究也发现,B.pseudomallei感染肺上皮细胞A549后,自噬水平较未感染组明显减弱;通过m RNA表达谱芯片筛选发现自噬相关基因ATG10下调最显著。那么,B.pseudomallei是如何调控及抑制自噬,以利于其在宿主细胞中的存活和复制,使得机体不能有效清除B.pseudomallei,从而导致持续性感染的呢?mi RNAs作为病原与宿主相互作用的信号调节分子,可通过介导清除病原体的自噬反应,发挥抗病原微生物的感染免疫调节作用。为了探究B.pseudomallei感染过程中调控自噬的具体机制,以及mi RNAs是否参与、如何参与自噬调节的分子机制,我们以肺上皮细胞A549为细胞模型,开展了B.pseudomallei感染A549细胞后mi RNA表达谱芯片的筛选,对上调的mi RNA进行生物信息学分析,结果发现三种未被报道的mi RNAs:MIR4458、MIR4667-5p、MIR4668-5p,它们可在3个不同的感染时间点分别靶向自噬信号通路的关键分子ATG10,提示我们B.pseudomallei可能通过改变宿主细胞的mi RNAs表达谱,干扰宿主细胞自噬,实现逃避机体免疫清除的目的。因此,明确这些mi RNAs介导的自噬抑制在类鼻疽杆菌感染免疫逃逸中的机制,将有助于开发作用于mi RNAs或其靶标的新的治疗手段,尤其是针对类鼻疽杆菌此种耐药性强、胞内感染的病原,临床意义更为重要。【研究目的】1.明确B.pseudomallei感染A549细胞后自噬受抑的生物学现象;2.明确B.pseudomallei感染A549细胞后自噬相关蛋白ATG10的表达变化;3.探讨MIR4458、MIR4667-5p、MIR4668-5p负性调控自噬的具体机制以及对细胞清除胞内B.pseudomallei的影响;4.探讨B.pseudomallei感染对MIR4458、MIR4667-5p、MIR4668-5p转录调控的分子机制。【研究方法】1.B.pseudomallei感染A549细胞后对细胞自噬的影响首先建立B.pseudomallei感染A549细胞模型,通过透射电镜、Western blot、激光共聚焦检测B.pseudomallei感染A549细胞后细胞自噬水平变化情况;再者,药物激活或抑制自噬的条件下,通过细菌胞内计数实验评价自噬对A549细胞清除胞内B.pseudomallei能力的影响。2.B.pseudomallei感染A549细胞自噬相关基因ATG10下调特点研究通过m RNA表达谱芯片筛选,发现自噬相关蛋白ATG10下调最明显;结合表达谱芯片筛选结果,在m RNA和蛋白水平分析ATG10的时空表达特点;同时,构建ATG10真核表达质粒,回复下调的ATG10后,观察并检测自噬和细菌清除的变化。3.MIR4458、MIR4667-5p和MIR4668-5p通过ATG10介导抑制自噬的机制研究结合mi RNA表达谱芯片筛选和生物信息学预测分析,发现MIR4458、MIR4667-5p和MIR4668-5p可靶向自噬相关基因ATG10;首先构建荧光素酶报告载体进一步鉴定ATG10是MIR4458、MIR4667-5p和MIR4668-5p的靶基因;分别过表达或抑制MIR4458、MIR4667-5p和MIR4668-5p,通过Western blot、激光共聚焦以及胞内细菌计数实验观察mi RNAs对自噬和细菌清除的影响。4.B.pseudomallei感染A549细胞后上调MIR4458、MIR4667-5p、MIR4668-5p表达的转录调控分子机制研究利用UCSC生物信息学软件分析和预测MIR4458、MIR4667-5p、MIR4668-5p启动子区域的转录调控元件,发现均含有较大的Cp G岛;首先通过BSP和DNMT活性检测,观察B.pseudomallei感染后MIR4458、MIR4667-5p和MIR4668-5p的Cp G岛甲基化水平改变;再者,利用去甲基化药物5-Aza-Cd R处理,通过MSP和q RT-PCR实验,检测降低甲基化水平对MIR4458、MIR4667-5p和MIR4668-5p表达的影响。【研究结果】1.B.pseudomallei感染A549细胞后自噬受抑通过透射电镜、Western blot、激光共聚焦实验,发现B.pseudomallei感染A549细胞后自噬受抑;另外,在诱导或抑制自噬的条件下,通过胞内细菌计数实验发现激活自噬可以增强宿主细胞对B.pseudomallei的胞内清除能力。2.B.pseudomallei通过下调ATG10抑制自噬通过m RNA表达谱芯片筛选,发现B.pseudomallei感染后自噬相关蛋白ATG10下调最明显;利用q RT-PCR和Western blot实验分析,ATG10在m RNA和蛋白水平上均发生明显下调;同时,过表达ATG10可增强细胞自噬以及对B.pseudomallei的胞内清除能力。3.B.pseudomallei通过上调MIR4458、MIR4667-5p、MIR4668-5p抑制自噬通过生物信息学分析以及双荧光素酶报告实验、Western blot及q RT-PCR实验,预测并鉴定了ATG10是MIR4458、MIR4667-5p、MIR4668-5p的靶基因;分别过表达或抑制这3个mi RNA后,发现MIR4458、MIR4667-5p和MIR4668-5p通过ATG10介导了抑制宿主细胞自噬以及对B.pseudomallei胞内清除能力。4.B.pseudomallei感染后引起的MIR4458、MIR4667-5p、MIR4668-5p启动子区域Cp G岛甲基化水平降低与其表达调控有关通过BSP和DNMT活性检测,发现B.pseudomallei感染后MIR4458、MIR4667-5p和MIR4668-5p的Cp G岛甲基化水平降低;同时,去甲基化药物5-Aza-Cd R处理后,MSP分析MIR4458、MIR4667-5p和MIR4668-5p的Cp G岛发生去甲基化,进一步q RT-PCR检测发现MIR4458、MIR4667-5p和MIR4668-5p的表达均增加。【主要结论】B.pseudomallei感染可通过甲基化调控方式,诱导MIR4458、MIR4667-5p和MIR4668-5p的上调,进而实时、连续的抑制其靶基因ATG10的表达,负性调控自噬,从而影响宿主细胞对B.pseudomallei的清除,促进其胞内存活。【研究意义】本文的研究展示了一个新的调控自噬的机制,其创新性主要在于探讨了“B.pseudomallei、mi RNA与自噬”的作用模式,以mi RNA为切入点,初步阐明了B.pseudomallei干扰宿主细胞自噬,实现逃避宿主免疫清除的分子机制。该研究为类鼻疽杆菌感染的治疗提供了一个新的治疗靶标,同时也为其他胞内病原感染的治疗提供了借鉴。
[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�糓IR4458,MIR4667-5p鍜孧IR4668-5p鐨勪笂璋，

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