GEF-H1在抗结核免疫应答中的功能研究

发布时间：2018-06-05 15:37

本文选题：MTB + GEF-H1　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景结核病(tuberculosis,TB)是由结核分枝杆菌(Mycobacterium tuberculosis,MTB)感染引起的、对人类公共健康危害最大的传染病之一。据世界卫生组织(World Health Organization,WHO)统计,2015年新发结核病例1040万,其中48万为多重耐药结核,10万人为利福平耐药结核;结核致死人数高达140万,约19万人死于多重耐药结核。由此可见,结核病对人类的生命健康威胁仍然十分严峻,是亟待解决的全球性公共卫生问题。抗生素长期使用以及抗结核药物使用的不合理,导致耐多药结核菌株的产生和传播;结核-HIV的联合感染,使结核病的治疗变得更加复杂和困难。深入研究MTB致病机制、免疫病理及免疫系统杀伤和清除MTB的机制,对寻找有效的抗结核治疗和预防的新靶点,具有非常重要的意义。机体的免疫系统防御反应在抵抗MTB感染中发挥着至关重要的作用。MTB被肺泡和间质巨噬细胞、以及局部树突状细胞(dendritic cell,DC)识别时,引发先天免疫应答。巨噬细胞(macrophage,Mφ)是机体抗结核感染的第一道防线,通过表达Toll样受体(Toll-like receptor,TLR)、免疫球蛋白受体、补体受体和凝集素受体等多种模式识别受体(pattern recognition receptors,PRR),与入侵的MTB相关抗原结合,从而介导MTB的粘附及吞噬,发挥胞内杀菌效应。Ras同源家族的小鸟嘌呤核苷酸结合蛋白(The Rho family of small GTP-binding proteins,Rho-GTPases)作为基本细胞活动的分子开关,通过结合GTP或GDP而发生活性与非活性状态的改变,在肌动蛋白细胞骨架重组、细胞迁移和粘附、活性氧形成和细胞凋亡中等多种细胞生命过程中发挥着至关重要的调控作用。鸟嘌呤核苷酸交换因子(guanine nucleotide exchange factor,GEF)家族成员促进GTP与GDP交换,是确保Rho-GTPases在特定空间和时间激活的理想分子。在GEF家族中,鸟嘌呤核苷酸交换因子H1(GEF-H1)活性受微管结合的调节,从而参与细胞形变、p53肿瘤抑制基因的突变、细胞周期等生理生化过程。研究表明,GEF-H1的表达缺失或上调可调节细胞信号转导、Rho家族成员之一RhoA活性及抗感染免疫反应。然而,目前尚未见到GEF-H1调控被感染Mφ抵抗MTB感染的相关报道。本课题探究了 MTB感染Mφ后GEF-H1的表达变化及调节机制,并探讨了GEF-H1通过调控p38 MAPK、TBK1通路,调节MTB感染Mφ中促炎因子表达,最终影响Mφ抑菌活性的机制,为进一步研究GEF-H1在Mφ抗结核感染免疫中的作用机制奠定了基础,为开发通过GEF-H1调控Mφ抑菌活性的抗结核药物提供了潜在靶点,对结核的有效防控具有重要意义。研究目的1.确定GEF-H1与MTB感染的相关性,阐明MTB感染如何诱导GEF-H1表达;2.揭示GEF-H1对Mφ清除MTB的调控作用;研究方法1.检测MTB感染后GEF-H1的表达水平a.采用采用实时荧光定量PCR(Real-time PCR)检测MTB感染Mφ中GEF-H1的mRNA表达水平;b.采用Western blot,检测MTB感染Mφ中GEF-H1的蛋白表达水平。2.研究MTB感染诱导GEF-H1表达增加的机制a.采用多种信号通路抑制剂预处理Mφ后,Real-time PCR检测Mφ中GEF-H1的mRNA表达水平;b.免疫荧光-激光扫描共聚焦显微镜观察MTB感染Mφ中,GEF-H1与微管的共定位。3.探讨调节Mφ炎症反应及抑菌效应的分子机制a.采用siRNA沉默Mφ中GEF-H1的表达;b.通过平板菌落计数(CFU)实验,检测沉默GEF-H1后MTB感染Mφ的抑菌活性;c.采用流式细胞术检测Mφ吞噬Texas-Red标记BCG的能力;d.Griess法和Western blot检测沉默GEF-H1后MTB感染Mφ的NO产生和自噬发生情况;e.用Real-time PCR和ELISA检测GEF-H1表达沉默对MTB感染Mφ促炎因子表达的调节效应;f.Western blot检测沉默GEF-H1 后MTB感染Mφ的p38 MAPK、TBK1 通路活化;g.G-LISA检测沉默GEF-H1后MTB感染Mφ的RhoA活性。研究结果1.MTB感染Mφ细胞系后,GEF-H1的表达水平显著上调,并呈时间及感染剂量依赖性。2.MTB感染诱导的GEF-H1高表达依赖于p38、ERK、JNK及NF-κB信号通路,同时与MTB感染后微管的解聚有关。3.MTB感染Mφ后,GEF-H1促进Mφ吞噬及清除胞内MTB。4.MTB感染Mφ后,GEF-H1通过激活p38MAPK和TBK1信号通路促进Mφ表达与分泌促炎细胞因子IL-6、IL-1β,从而发挥抑菌活性,且该调控效应不依赖于RhoA的激活。5.GEF-H1的抑菌活性并不依赖MTB感染后Mφ中NO的产生以及自噬的发生。结论本研究发现MTB感染诱导GEF-H1表达水平的显著上调,并进一步在体外MTB感染Mφ模型中揭示,GEF-H1通过活化p38和TBK1信号通路,促进Mφ表达与分泌促炎因子,继而促进Mφ吞噬并清除胞内MTB的新机制。本研究为寻找结核病治疗的新靶点,开发新的防治措施提供了理论依据。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R52

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