结核分枝杆菌mce1操纵子上Rv0177蛋白与巨噬细胞互作机制的研究

发布时间：2018-03-09 01:25

本文选题：结核分枝杆菌　切入点：Rv0177　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：致病性分枝杆菌主要包括结核分枝杆菌(M.tuberculosis)、牛分枝杆菌(M.bovis)、麻风分枝杆菌(M.leprae)。其中,结核分枝杆菌是引起结核病的主要病原菌,它可侵犯人类全身各器官,但主要以肺部为主。根据疾病控制和预防中心的数据,2015年有180万人因罹患结核病而丧失生命,世界上仍有1/3的人口感染结核菌。近年来,随着抗生素广泛和大量地使用,多重耐药结核分枝杆菌(mutiple-drug resistant tuberculosis)和广泛耐药结核分枝杆菌(extensive-drug resistant tuberculosis)剧增,这使结核病的治疗变得更加困难。另外,新研究发现非结核分枝杆菌(nontuberculosis mycobacteria,NTM)(指除了结核分枝杆菌、牛分枝杆菌和麻风分枝杆菌以外的分枝杆菌)也可以引起肺部以及肺部以外组织器官的严重病变。因此,研究和揭示结核分枝杆菌的致病机理和机体的免疫保护机制为预防和治疗结核病提供有价值的研究基础显的尤为重要。Rv0177是位于13个基因共转录的mce1操纵子上的一个保守假定蛋白。根据文章报道,该蛋白是结核分枝杆菌胞内存活所必需,宿主细胞中上调表达,且在人类和人类肠道微生物中没有其同源蛋白的存在。因此,我们预测它可能参与结核分枝杆菌的毒力,是一个有效的药物靶标。在本实验中,我们构建了pNIT_rv0177重组质粒,将pNIT空质粒和pNIT_rv0177重组质粒分别电转入非致病性的耻垢分枝杆菌(M.smegmatis)中。我们通过特异性引物对电转的单菌落进行PCR扩增和进一步的Western blotting方法验证Rv0177蛋白在耻垢分枝杆菌中的成功构建和表达。我们通过离心分离实验,证明了Rv0177蛋白定位在耻垢分枝杆菌的细胞壁上。我们发现MS_Rv0177和MS_Vec菌株在对数期的生长速率是一样的。MS_Rv0177相比于空载菌MS_Vec生物膜的形成更为光滑;菌株的滑动能力显著增强;透射电镜分析发现过表达菌株相比于空载菌的胞内“空泡”要少,细胞壁的表面更紧致严密;溴化乙锭小分子渗透性检测也发现了过表达菌株显著地降低了细胞壁的通透性。另外,在体外低酸、氧化压力条件的培养下,我们发现MS_Rv0177过表达菌株在酸性培养基和双氧水氧化压力条件下具有一定的生长耐受性。然而,我们通过提取MS_Rv0177和MS_Vec重组菌的总量脂肪酸,进行GC-MS分析;提取两株重组菌细胞壁的肽聚糖脂类,进行薄层层析(TLC)分析;我们发现过表达菌株并没有明显地改变这些成分的含量。我们用培养诱导后的MS_Rv0177和MS_Vec分别去侵染小鼠RAW264.7细胞和人类的THP-1细胞,发现过表达菌株在以上两种巨噬细胞中存活敏感,并且发现MS_Rv0177对小鼠巨噬细胞的入侵能力与MS_Vec菌株相近。MS_Rv0177上调了巨噬细胞细胞因子MCP-1的表达,下调了IL-6细胞因子的表达;同时上调了MCPIP的表达,诱导了内质网压力伴侣蛋白CHOP的表达。我们通过MTT实验方法和细胞培养上清LDH活性检测法发现MS_Rv0177对小鼠巨噬细胞的存活有一定的影响,并且MS_Rv0177能够促进小鼠RAW264.7细胞的凋亡。同时,我们用NF-κB,JNK,p38的特异性信号路径抑制剂去处理侵染前的小鼠RAW264.7细胞,发现JNK信号通路抑制剂能够显著的抑制MS_Rv0177诱导的小鼠RAW264.7细胞中MCP-1,MCPIP和CHOP的转录;表明JNK信号路径在重组菌MS_Rv0177与小鼠RAW264.7细胞互作中扮演着重要的角色。
[Abstract]:Pathogenic mycobacteria including Mycobacterium tuberculosis (M.tuberculosis), Mycobacterium bovis (M.bovis), Mycobacterium leprae (M.leprae). Among them, Mycobacterium tuberculosis is the main pathogen of TB bacteria, it can be violated in various organs of the human body, but mainly in the lung. According to the Centers for Disease Control and prevention data. In 2015 1 million 800 thousand people suffering from tuberculosis and loss of life, the world still has a population of 1/3 infected with Mycobacterium tuberculosis. In recent years, with the extensive use of antibiotics and large, multi drug resistant Mycobacterium tuberculosis (mutiple-drug resistant tuberculosis) and extensively drug-resistant Mycobacterium tuberculosis (extensive-drug resistant tuberculosis) this increase, the TB treatment becomes more difficult. In addition, the new study found that non Mycobacterium tuberculosis (nontuberculosis mycobacteria, NTM) (except Mycobacterium tuberculosis, Mycobacterium bovis and Ma Outside the wind of Mycobacterium Mycobacterium) can also cause severe lesions outside the lungs and lung tissues and organs. Therefore, the protective mechanism of pathogenic mechanism and reveal the body of Mycobacterium tuberculosis is particularly important in.Rv0177 prevention and treatment of tuberculosis to provide the basic research value obviously is a conserved hypothetical protein in 13 genes a total of transcription mce1 operon. According to the article, the protein of Mycobacterium tuberculosis is essential for cell survival, expression in the host cell, and not the same source of protein in human and human intestinal microflora in existence. Therefore, we predict that it may be involved in the virulence of Mycobacterium tuberculosis, is an effective drug target. In this experiment, we constructed the recombinant plasmid of pNIT_rv0177, pNIT and pNIT_rv0177 empty plasmid recombinant plasmids were electroporated into non pathogenic Mycobacterium smegmatis Coli (M.smegmatis). And the expression of single colony through our specific primers electrotransfer of Rv0177 protein was amplified by PCR and verified further by Western blotting method in Mycobacterium smegmatis. We successfully constructed by centrifugal separation experiments, proved that the Rv0177 protein located in the cell wall of Mycobacterium smegmatis. We found that MS_Rv0177 and MS_Vec strains in the growth rate of the logarithmic phase is formed as compared to.MS_Rv0177 MS_Vec biofilm bacteria load is more smooth; significantly enhance the sliding ability of strains; transmission electron microscopy analysis showed that over expression strain compared to the empty bacteria intracellular vacuole to less cell wall surface more compact tight; small ethidium bromide molecular permeability testing also found that over expression strain significantly decreased the permeability of cell wall. In addition, the in vitro culture of low acid, oxidative stress conditions, we found that MS Overexpression of _Rv0177 strains with the growth of a certain degree of tolerance in acidic medium and hydrogen peroxide under pressure. However, we extract MS_Rv0177 and MS_Vec recombinant strains in total fatty acids, GC-MS analysis; from two strains of recombinant bacterial cells wall peptidoglycan glycolipids, by thin-layer chromatography (TLC) analysis we found; the content of over expression strain did not significantly change these components. We use the training after the induction of MS_Rv0177 and MS_Vec respectively to infect mouse RAW264.7 cells and human THP-1 cells, overexpression strains in the above two kinds of macrophage survival sensitive, and found that MS_Rv0177 on mouse macrophage invasion ability and strain MS_Vec similar up regulation of.MS_Rv0177 the expression of macrophage cytokine MCP-1, down regulated the expression of IL-6 cytokines; also up-regulated the expression of MCPIP, induced endoplasmic reticulum stress partner egg The expression of white CHOP. We through the MTT experiment method and cell culture method was used to detect the supernatant LDH activity found MS_Rv0177 on macrophage survival has certain effect, and MS_Rv0177 can promote the apoptosis of mouse RAW264.7 cells. At the same time, we use the NF- kappa B, JNK, a specific inhibitor of p38 signal path to infect mice before RAW264.7 cells, found that JNK signaling pathway inhibitor MCP-1 can significantly inhibit MS_Rv0177 induced RAW264.7 cells, the transcription of MCPIP and CHOP; that JNK signal pathway plays an important role in recombinant MS_Rv0177 and mouse RAW264.7 cell interaction.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378.911

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