本文選題：結(jié)核分枝桿菌　切入點(diǎn)：Rv0177　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：致病性分枝桿菌主要包括結(jié)核分枝桿菌(M.tuberculosis)、牛分枝桿菌(M.bovis)、麻風(fēng)分枝桿菌(M.leprae)。其中,結(jié)核分枝桿菌是引起結(jié)核病的主要病原菌,它可侵犯人類全身各器官,但主要以肺部為主。根據(jù)疾病控制和預(yù)防中心的數(shù)據(jù),2015年有180萬(wàn)人因罹患結(jié)核病而喪失生命,世界上仍有1/3的人口感染結(jié)核菌。近年來(lái),隨著抗生素廣泛和大量地使用,多重耐藥結(jié)核分枝桿菌(mutiple-drug resistant tuberculosis)和廣泛耐藥結(jié)核分枝桿菌(extensive-drug resistant tuberculosis)劇增,這使結(jié)核病的治療變得更加困難。另外,新研究發(fā)現(xiàn)非結(jié)核分枝桿菌(nontuberculosis mycobacteria,NTM)(指除了結(jié)核分枝桿菌、牛分枝桿菌和麻風(fēng)分枝桿菌以外的分枝桿菌)也可以引起肺部以及肺部以外組織器官的嚴(yán)重病變。因此,研究和揭示結(jié)核分枝桿菌的致病機(jī)理和機(jī)體的免疫保護(hù)機(jī)制為預(yù)防和治療結(jié)核病提供有價(jià)值的研究基礎(chǔ)顯的尤為重要。Rv0177是位于13個(gè)基因共轉(zhuǎn)錄的mce1操縱子上的一個(gè)保守假定蛋白。根據(jù)文章報(bào)道,該蛋白是結(jié)核分枝桿菌胞內(nèi)存活所必需,宿主細(xì)胞中上調(diào)表達(dá),且在人類和人類腸道微生物中沒(méi)有其同源蛋白的存在。因此,我們預(yù)測(cè)它可能參與結(jié)核分枝桿菌的毒力,是一個(gè)有效的藥物靶標(biāo)。在本實(shí)驗(yàn)中,我們構(gòu)建了pNIT_rv0177重組質(zhì)粒,將pNIT空質(zhì)粒和pNIT_rv0177重組質(zhì)粒分別電轉(zhuǎn)入非致病性的恥垢分枝桿菌(M.smegmatis)中。我們通過(guò)特異性引物對(duì)電轉(zhuǎn)的單菌落進(jìn)行PCR擴(kuò)增和進(jìn)一步的Western blotting方法驗(yàn)證Rv0177蛋白在恥垢分枝桿菌中的成功構(gòu)建和表達(dá)。我們通過(guò)離心分離實(shí)驗(yàn),證明了Rv0177蛋白定位在恥垢分枝桿菌的細(xì)胞壁上。我們發(fā)現(xiàn)MS_Rv0177和MS_Vec菌株在對(duì)數(shù)期的生長(zhǎng)速率是一樣的。MS_Rv0177相比于空載菌MS_Vec生物膜的形成更為光滑;菌株的滑動(dòng)能力顯著增強(qiáng);透射電鏡分析發(fā)現(xiàn)過(guò)表達(dá)菌株相比于空載菌的胞內(nèi)“空泡”要少,細(xì)胞壁的表面更緊致嚴(yán)密;溴化乙錠小分子滲透性檢測(cè)也發(fā)現(xiàn)了過(guò)表達(dá)菌株顯著地降低了細(xì)胞壁的通透性。另外,在體外低酸、氧化壓力條件的培養(yǎng)下,我們發(fā)現(xiàn)MS_Rv0177過(guò)表達(dá)菌株在酸性培養(yǎng)基和雙氧水氧化壓力條件下具有一定的生長(zhǎng)耐受性。然而,我們通過(guò)提取MS_Rv0177和MS_Vec重組菌的總量脂肪酸,進(jìn)行GC-MS分析;提取兩株重組菌細(xì)胞壁的肽聚糖脂類,進(jìn)行薄層層析(TLC)分析;我們發(fā)現(xiàn)過(guò)表達(dá)菌株并沒(méi)有明顯地改變這些成分的含量。我們用培養(yǎng)誘導(dǎo)后的MS_Rv0177和MS_Vec分別去侵染小鼠RAW264.7細(xì)胞和人類的THP-1細(xì)胞,發(fā)現(xiàn)過(guò)表達(dá)菌株在以上兩種巨噬細(xì)胞中存活敏感,并且發(fā)現(xiàn)MS_Rv0177對(duì)小鼠巨噬細(xì)胞的入侵能力與MS_Vec菌株相近。MS_Rv0177上調(diào)了巨噬細(xì)胞細(xì)胞因子MCP-1的表達(dá),下調(diào)了IL-6細(xì)胞因子的表達(dá);同時(shí)上調(diào)了MCPIP的表達(dá),誘導(dǎo)了內(nèi)質(zhì)網(wǎng)壓力伴侶蛋白CHOP的表達(dá)。我們通過(guò)MTT實(shí)驗(yàn)方法和細(xì)胞培養(yǎng)上清LDH活性檢測(cè)法發(fā)現(xiàn)MS_Rv0177對(duì)小鼠巨噬細(xì)胞的存活有一定的影響,并且MS_Rv0177能夠促進(jìn)小鼠RAW264.7細(xì)胞的凋亡。同時(shí),我們用NF-κB,JNK,p38的特異性信號(hào)路徑抑制劑去處理侵染前的小鼠RAW264.7細(xì)胞,發(fā)現(xiàn)JNK信號(hào)通路抑制劑能夠顯著的抑制MS_Rv0177誘導(dǎo)的小鼠RAW264.7細(xì)胞中MCP-1,MCPIP和CHOP的轉(zhuǎn)錄;表明JNK信號(hào)路徑在重組菌MS_Rv0177與小鼠RAW264.7細(xì)胞互作中扮演著重要的角色。
[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.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R378.911

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相關(guān)期刊論文 前1條

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本文編號(hào)：1586410