本文選題：MTB + GEF-H1��； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景結(jié)核病(tuberculosis,TB)是由結(jié)核分枝桿菌(Mycobacterium tuberculosis,MTB)感染引起的、對(duì)人類公共健康危害最大的傳染病之一。據(jù)世界衛(wèi)生組織(World Health Organization,WHO)統(tǒng)計(jì),2015年新發(fā)結(jié)核病例1040萬(wàn),其中48萬(wàn)為多重耐藥結(jié)核,10萬(wàn)人為利福平耐藥結(jié)核;結(jié)核致死人數(shù)高達(dá)140萬(wàn),約19萬(wàn)人死于多重耐藥結(jié)核。由此可見(jiàn),結(jié)核病對(duì)人類的生命健康威脅仍然十分嚴(yán)峻,是亟待解決的全球性公共衛(wèi)生問(wèn)題�？股亻L(zhǎng)期使用以及抗結(jié)核藥物使用的不合理,導(dǎo)致耐多藥結(jié)核菌株的產(chǎn)生和傳播;結(jié)核-HIV的聯(lián)合感染,使結(jié)核病的治療變得更加復(fù)雜和困難。深入研究MTB致病機(jī)制、免疫病理及免疫系統(tǒng)殺傷和清除MTB的機(jī)制,對(duì)尋找有效的抗結(jié)核治療和預(yù)防的新靶點(diǎn),具有非常重要的意義。機(jī)體的免疫系統(tǒng)防御反應(yīng)在抵抗MTB感染中發(fā)揮著至關(guān)重要的作用。MTB被肺泡和間質(zhì)巨噬細(xì)胞、以及局部樹(shù)突狀細(xì)胞(dendritic cell,DC)識(shí)別時(shí),引發(fā)先天免疫應(yīng)答。巨噬細(xì)胞(macrophage,Mφ)是機(jī)體抗結(jié)核感染的第一道防線,通過(guò)表達(dá)Toll樣受體(Toll-like receptor,TLR)、免疫球蛋白受體、補(bǔ)體受體和凝集素受體等多種模式識(shí)別受體(pattern recognition receptors,PRR),與入侵的MTB相關(guān)抗原結(jié)合,從而介導(dǎo)MTB的粘附及吞噬,發(fā)揮胞內(nèi)殺菌效應(yīng)。Ras同源家族的小鳥(niǎo)嘌呤核苷酸結(jié)合蛋白(The Rho family of small GTP-binding proteins,Rho-GTPases)作為基本細(xì)胞活動(dòng)的分子開(kāi)關(guān),通過(guò)結(jié)合GTP或GDP而發(fā)生活性與非活性狀態(tài)的改變,在肌動(dòng)蛋白細(xì)胞骨架重組、細(xì)胞遷移和粘附、活性氧形成和細(xì)胞凋亡中等多種細(xì)胞生命過(guò)程中發(fā)揮著至關(guān)重要的調(diào)控作用。鳥(niǎo)嘌呤核苷酸交換因子(guanine nucleotide exchange factor,GEF)家族成員促進(jìn)GTP與GDP交換,是確保Rho-GTPases在特定空間和時(shí)間激活的理想分子。在GEF家族中,鳥(niǎo)嘌呤核苷酸交換因子H1(GEF-H1)活性受微管結(jié)合的調(diào)節(jié),從而參與細(xì)胞形變、p53腫瘤抑制基因的突變、細(xì)胞周期等生理生化過(guò)程。研究表明,GEF-H1的表達(dá)缺失或上調(diào)可調(diào)節(jié)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)、Rho家族成員之一RhoA活性及抗感染免疫反應(yīng)。然而,目前尚未見(jiàn)到GEF-H1調(diào)控被感染Mφ抵抗MTB感染的相關(guān)報(bào)道。本課題探究了 MTB感染Mφ后GEF-H1的表達(dá)變化及調(diào)節(jié)機(jī)制,并探討了GEF-H1通過(guò)調(diào)控p38 MAPK、TBK1通路,調(diào)節(jié)MTB感染Mφ中促炎因子表達(dá),最終影響Mφ抑菌活性的機(jī)制,為進(jìn)一步研究GEF-H1在Mφ抗結(jié)核感染免疫中的作用機(jī)制奠定了基礎(chǔ),為開(kāi)發(fā)通過(guò)GEF-H1調(diào)控Mφ抑菌活性的抗結(jié)核藥物提供了潛在靶點(diǎn),對(duì)結(jié)核的有效防控具有重要意義。研究目的1.確定GEF-H1與MTB感染的相關(guān)性,闡明MTB感染如何誘導(dǎo)GEF-H1表達(dá);2.揭示GEF-H1對(duì)Mφ清除MTB的調(diào)控作用;研究方法1.檢測(cè)MTB感染后GEF-H1的表達(dá)水平a.采用采用實(shí)時(shí)熒光定量PCR(Real-time PCR)檢測(cè)MTB感染Mφ中GEF-H1的mRNA表達(dá)水平;b.采用Western blot,檢測(cè)MTB感染Mφ中GEF-H1的蛋白表達(dá)水平。2.研究MTB感染誘導(dǎo)GEF-H1表達(dá)增加的機(jī)制a.采用多種信號(hào)通路抑制劑預(yù)處理Mφ后,Real-time PCR檢測(cè)Mφ中GEF-H1的mRNA表達(dá)水平;b.免疫熒光-激光掃描共聚焦顯微鏡觀察MTB感染Mφ中,GEF-H1與微管的共定位。3.探討調(diào)節(jié)Mφ炎癥反應(yīng)及抑菌效應(yīng)的分子機(jī)制a.采用siRNA沉默Mφ中GEF-H1的表達(dá);b.通過(guò)平板菌落計(jì)數(shù)(CFU)實(shí)驗(yàn),檢測(cè)沉默GEF-H1后MTB感染Mφ的抑菌活性;c.采用流式細(xì)胞術(shù)檢測(cè)Mφ吞噬Texas-Red標(biāo)記BCG的能力;d.Griess法和Western blot檢測(cè)沉默GEF-H1后MTB感染Mφ的NO產(chǎn)生和自噬發(fā)生情況;e.用Real-time PCR和ELISA檢測(cè)GEF-H1表達(dá)沉默對(duì)MTB感染Mφ促炎因子表達(dá)的調(diào)節(jié)效應(yīng);f.Western blot檢測(cè)沉默GEF-H1 后MTB感染Mφ的p38 MAPK、TBK1 通路活化;g.G-LISA檢測(cè)沉默GEF-H1后MTB感染Mφ的RhoA活性。研究結(jié)果1.MTB感染Mφ細(xì)胞系后,GEF-H1的表達(dá)水平顯著上調(diào),并呈時(shí)間及感染劑量依賴性。2.MTB感染誘導(dǎo)的GEF-H1高表達(dá)依賴于p38、ERK、JNK及NF-κB信號(hào)通路,同時(shí)與MTB感染后微管的解聚有關(guān)。3.MTB感染Mφ后,GEF-H1促進(jìn)Mφ吞噬及清除胞內(nèi)MTB。4.MTB感染Mφ后,GEF-H1通過(guò)激活p38MAPK和TBK1信號(hào)通路促進(jìn)Mφ表達(dá)與分泌促炎細(xì)胞因子IL-6、IL-1β,從而發(fā)揮抑菌活性,且該調(diào)控效應(yīng)不依賴于RhoA的激活。5.GEF-H1的抑菌活性并不依賴MTB感染后Mφ中NO的產(chǎn)生以及自噬的發(fā)生。結(jié)論本研究發(fā)現(xiàn)MTB感染誘導(dǎo)GEF-H1表達(dá)水平的顯著上調(diào),并進(jìn)一步在體外MTB感染Mφ模型中揭示,GEF-H1通過(guò)活化p38和TBK1信號(hào)通路,促進(jìn)Mφ表達(dá)與分泌促炎因子,繼而促進(jìn)Mφ吞噬并清除胞內(nèi)MTB的新機(jī)制。本研究為尋找結(jié)核病治療的新靶點(diǎn),開(kāi)發(fā)新的防治措施提供了理論依據(jù)。
[Abstract]:Background tuberculosis (TB) is one of the most dangerous infectious diseases caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) infection. According to the WHO (World Health Organization, WHO) statistics, 10 million 400 thousand new tuberculosis cases were reported in 2015, of which 480 thousand were multidrug-resistant tuberculosis and 100 thousand people were multidrug-resistant. The number of rifampicin resistant tuberculosis; the number of deaths from tuberculosis to 1 million 400 thousand and about 190 thousand people died of multidrug-resistant tuberculosis. This shows that tuberculosis is still a serious threat to human life and health. It is a global public health problem to be solved urgently. The long-term use of antibiotics and the irrational use of anti tuberculosis drugs lead to multi drug resistant strains. The joint infection of tuberculosis -HIV makes the treatment of tuberculosis more complicated and difficult. It is of great importance to study the pathogenesis of MTB, the mechanism of immune pathology and the killing of the immune system and the elimination of MTB. It is of great importance to find effective targets for anti tuberculosis treatment and prevention. The immune system defense response of the body is resistant to resistance. MTB infection plays a vital role in.MTB by the identification of alveolar macrophages and interstitial macrophages and dendritic cell (DC), which causes the innate immune response. Macrophages (macrophage, M phi) are the first line of defense against tuberculosis infection, by expressing the Toll like receptor (Toll-like receptor, TLR), immunoglobulin Body, complement receptor and agglutinin receptor, such as pattern recognition receptors (PRR), combined with the intrusions of MTB associated antigen, thus mediate the adhesion and phagocytosis of MTB, and play the intracellular bactericidal effect of the.Ras homologous family of the bird purine nucleotide binding protein (The Rho family of small) As a molecular switch for basic cell activity, the changes in life and inactivity by combining GTP or GDP play a crucial role in the regulatory role of actin cytoskeleton reorganization, cell migration and adhesion, reactive oxygen species formation and cell apoptosis. Guanine nucleotide exchange factor (guanine) Nucleotide exchange factor, GEF) family members promote the exchange of GTP with GDP, is an ideal molecule to ensure Rho-GTPases activation in a specific space and time. In the GEF family, the H1 (GEF-H1) activity of guanine nucleotide exchange factor is regulated by microtubule binding, thus participating in cell transformation, p53 tumor suppressor gene mutation, cell cycle and so on. The study showed that the expression deletion or up-regulation of GEF-H1 can regulate cell signal transduction, RhoA activity and anti infection immune response of Rho family members. However, there has not been a report on the regulation of M Phi resistance to MTB infection by GEF-H1. This subject explores the expression and regulation mechanism of GEF-H1 after MTB infection M. GEF-H1 regulates the expression of pro-inflammatory factors in MTB infection M Phi by regulating p38 MAPK and TBK1 pathway, and ultimately affects the mechanism of M Phi inhibitory activity. It lays the foundation for further research on the mechanism of GEF-H1 in the anti tuberculosis infection immunity of M phi, and provides potential targets for developing anti tuberculosis drugs by GEF-H1 regulating M Phi activity, and for tuberculosis. Effective prevention and control is of great significance. 1. the study is to determine the correlation between GEF-H1 and MTB infection, to clarify how MTB infection induces GEF-H1 expression, and 2. to reveal the regulatory role of GEF-H1 on M Phi clearance of MTB; method 1. to detect the expression level of GEF-H1 after MTB infection (a.) The expression level of mRNA, B. using Western blot, detection of the protein expression level of GEF-H1 in MTB infection M phi, the mechanism of MTB infection induced GEF-H1 expression increased, A. using a variety of signal pathway inhibitors preprocessing M [Phi]. The co localization of GEF-H1 and microtubule in M Phi was used to investigate the molecular mechanism of regulating the inflammatory response and bacteriostasis effect of M phi, A. used siRNA to silence the expression of GEF-H1 in M phi; B. through the flat colony count (CFU) test was used to detect the bacteriostasis activity of MTB infection. The detection of NO production and autophagy of M Phi after MTB infection after GEF-H1 was detected by method and Western blot; e. used Real-time PCR and ELISA to detect the regulation effect of GEF-H1 expression of silence on the expression of MTB infection Phi factor. The RhoA activity of M [Phi]. After 1.MTB infection, the expression level of GEF-H1 was significantly up-regulated, and the time and dose dependent.2.MTB infection induced GEF-H1 high expression depended on p38, ERK, JNK and NF- kappa B signal pathway. After MTB.4.MTB infection of M phi, GEF-H1 promotes M Phi expression and secretion of pro-inflammatory cytokine IL-6, IL-1 beta, by activating p38MAPK and TBK1 signaling pathway, thus exerting bacteriostasis activity, and this regulation effect is not dependent on the inhibitory activity of RhoA activated.5.GEF-H1 without dependence on the production of M [M] and the occurrence of autophagy after MTB infection. MTB infection induced the significant up-regulation of GEF-H1 expression level, and further revealed in the M Phi model of MTB infection in vitro, GEF-H1 promotes the expression and secretion of proinflammatory cytokines by activating p38 and TBK1 signaling pathways, and then promotes the new mechanism of M phagocytosis and clearance of intracellular MTB. This study is to develop new preventive measures for finding new targets for tuberculosis treatment. The theoretical basis is provided.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R52

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Ameer Khusro;Chirom Aarti;Paul Agastian;;Anti-tubercular peptides:A quest of future therapeutic weapon to combat tuberculosis[J];Asian Pacific Journal of Tropical Medicine;2016年11期

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本文編號(hào)：1982494