TLR9基因遗传多态性与疾病关系的研究进展

近年来对TLRs的研究主要集中在人类的TLRs的遗传多态性在各种疾病中的免疫学作用等方面的研究。对其他哺乳动物TLRs的免疫学研究还不是很多。本文综述TLR9基因的遗传多态性与几种疾病关系的研究最新进展。     

DNA的识别分子及其识别模式

固有免疫以TLR9依赖和TLR9非依赖的方式对DNA识别,在保护性免疫和病理性自身免疫反应中发挥重要作用。目前发现TLR9,DAI（DLM-1／ZBPl）,RIG—I,尚未定义的识别B—DNA的分子,损伤DNA结合分子如：DNA—PKCS,Ku70,p53,ATM,ATR和含有SAP或PHD结构域等的分子可以识别DNA,并且分别以不同的识别模式识别细胞不同部位或不同类型的DNA。近几年来国内外针对DNA识别的研究较多并取得了重要进展,给研究保护性免疫和自身免疫提供了新的视野。文中结合作者所在实验室的研究探索,对DNA的识别分子和识别模式的研究进展进行综述。     

去泛素化酶7与髓样分化因子88蛋白相互作用的研究

Toll-like受体（TLRs）介导的信号途径不仅参与机体识别病原微生物入侵、诱导免疫应答,而且还可诱导机体产生破坏性炎症反应．通过免疫共沉淀、免疫荧光共定位、间接免疫荧光等实验方法鉴定去泛素化酶7（USP7）和TLR信号途径中重要接头蛋白——髓样分化因子88（MyD88）的相互作用,结果表明USP7和MyD88可在细胞质中共定位并可发生相互作用．该研究对阐释TLR信号通路的机制奠定了重要的基础．     

LR2和TLR6在鸟分枝杆菌诱导巨噬细胞凋亡中的作用

[目的]建立鸟分枝杆菌感染巨噬细胞模型,探讨Toll样受体2(TLR2)和Toll样受体6(TLR6)在鸟分枝杆菌诱导巨噬细胞凋亡中的作用,为阐明鸟分枝杆菌致病机制提供依据。[方法]用鸟分枝杆菌感染U937细胞,于感染0 h、8 h、16 h、24 h和48 h后分别提取细胞总蛋白,并通过Western blot方法检测TLR2和TLR6的表达;分别提取细胞培养上清液,利用ELISA技术检测肿瘤坏死因子(TNF-α)含量的变化情况。用鸟分枝杆菌感染阻断TLR2和TLR6后的U937细胞,通过Western blot方法分别检测其促凋亡因子BAX和抗凋亡因子BCL-2的表达量,用ELISA技术检测细胞培养上清液中TNF-α含量变化情况,用流式细胞仪检测U937细胞的凋亡变化。[结果]鸟分枝杆菌感染U937细胞可引起TLR2、TLR6和TNF-α表达上调;用鸟分枝杆菌感染阻断TLR2和TLR6后的U937细胞,可使U937细胞内促凋亡因子BAX和TNF-α表达量下降(P<0.05),抗凋亡因子BCL-2表达量升高(P<0.05),并可以明显降低感染鸟分枝杆菌后的细胞的凋亡率(P<0.05)。[结论]TLR2和TLR6与巨噬细胞凋亡相关,巨噬细胞通过TLR2和TLR6来识别鸟分枝杆菌,而鸟分枝杆菌反过来通过促进TLR2和TLR6的表达来影响巨噬细胞相关凋亡通路,从而诱导巨噬细胞凋亡。     

斜带石斑鱼TLR5S基因结构及功能分析

Toll样受体5(Toll-like receptor 5,TLR5)属于先天免疫系统中的一种模式识别受体(PRR),可分为可溶型和跨膜型2种.它可以识别病原体相关分子模式(pathogen-associated molecular patterns,PAMP),从而启动信号转导.本研究从NCBI数据库中得到斜带石斑鱼(Epinephelus coioides)可溶型TLR5(ECTLR5S)cDNA全长序列(Genbank登录号:GQ396667).其cDNA序列全长2 439bp,包括69bp的5′UTR,435bp的3′UTR和1 935bp的开放阅读框,共编码644个氨基酸,预测编码蛋白质分子质量为72.28ku,等电点为6.37.将ECTLR5S基因氨基酸序列与其他脊椎动物的TLR5S基因氨基酸序列进行比对,结果显示出很高的相似性,其中与牙鲆相似度可达69%.Real time PCR检测结果显示ECTLR5S基因在斜带石斑鱼不同器官中均有表达,其中在肝脏中的表达量最高,其次是脾脏、血淋巴、肾脏等.Realtime PCR检测ECTLR5S基因在哈维氏弧菌(Vibrio harveyi)感染3,6,12,24,48h后的肝脏中表达情况,结果显示ECTLR5S基因在3,6,12,24h时表达量显著上调(p<0.05),而在48h下降至初始水平.这些结果说明ECTLR5S基因可能参与到斜带石斑鱼抗弧菌感染的免疫反应中,并且肝脏是其发挥作用的重要器官.     

老鼠和人对抗弓形虫感染的先天免疫应答研究概况

白细胞介素12(IL-12)和干扰素γ(IFN-γ)是弓形虫感染刺激机体先天防御系统所产生的两个关键性细胞因子,其信号是通过To1l样受体(TLR)介导的髓样分化因子(MyD88)依赖途径来激活早期的先天免疫反应.研究证实TLR 11在应答弓形虫感染过程中起着主要作用,但是它在人体中仅仅只是个假基因.因此,在缺失TLR11的情况下,人体受到弓形虫感染后如何引发先天性免疫和获得性免疫应答反应还需进一步研究.该文就人和老鼠体内对寄生虫防御起作用的感知分子和效应分子的相似性和区别进行探讨.     

电针对PCPA致失眠大鼠脾脏TLR7信号通路关键基因mRNA表达的影响

目的:观察电针对对氯苯丙氨酸(PCPA)致失眠大鼠脾脏Toll样受体7(TLR7)信号通路关键基因mRNA表达的影响,探讨电针治疗失眠的免疫学机制.方法:取健康SPF级Wistar雄性大鼠32只,随机分成空白组、模型组、电针组和安定组,每组8只.模型组以腹腔注射PCPA建立大鼠失眠模型,安定组给予腹腔注射安定,电针组给予电针神门(HT7)、三阴交(SP6)穴位,连续治疗5 d后用实时荧光定量PCR(RT-PCR)反应测定大鼠脾脏TLR7、髓样分化蛋白88(My D88),IL-1受体相关激酶4(IRAK4)、肿瘤坏死因子相关激酶6(TRAF6)、及核因子NF-κB(NF-κB)的mRNA表达.结果:与空白组对照,模型组TLR7、My D88、IRAK4及TRAF6的mRNA表达增加(P0.05);与模型组比较,电针组、安定组IRAK4、TRAF6及NF-k B的mRNA表达降低,电针组TLR7及My D88的mRNA表达降低(P0.05),但电针组与安定组比较,无统计学差异(P0.05).结论:失眠上调TLR7信号转导通路关键基因mRNA表达,TLR7可能参与免疫对睡眠的调节;电针可通过调节TLR7信号转导通路调控相关免疫物质的释放,改善睡眠及减轻失眠对机体带来的损伤.     

神经病理性疼痛的小胶质细胞机制

小胶质细胞是中枢神经系统内重要的免疫细胞,起递呈抗原、吞噬病原体、分泌多种细胞因子和神经修复的作用。神经损伤后,小胶质细胞会被激活,可释放大量的细胞因子、炎性介质,激活补体,引起神经炎症和神经免疫反应,导致各种神经功能紊乱,引起痛觉过敏和异常痛敏。本文从小胶质细胞的激活、以及激活途径的关键分子TLR4来探讨神经病理性疼痛的发病机制。     

CD9与免疫细胞、免疫因子相互作用及其功能的研究

CD9是Tetraspanins家族成员之一,属于白细胞分化抗原中的一种,它参与细胞黏附、运动、激活和分化等.近年有研究表明:CD9在激活T细胞、作为B细胞分子标记物以及诱导DC和MHC分子对抗原的提呈等方面都发挥着重要的作用.值得一提的是CD9在适应性免疫系统的作用可能追溯到原始的无颌类脊椎动物.对CD9与免疫细胞以及免疫因子的相互作用进行简要综述,为进一步理解和深入研究CD9在适应性免疫系统中所扮演的角色提供参考.     

凡纳对虾penaeidin2抗菌肽基因的克隆与性质研究

抗菌肽分子是无脊椎动物先天免疫系统中,直接发挥免疫效应的分子;它在对抗病原细菌侵染的过程中发挥着重要的功能.本文以凡纳对虾对虾素抗菌肽基因penaeidin2作为研究对象,系统地进行了基因克隆、氨基酸序列比对、分子进化关系以及分子结构的初步预测等研究.研究结果显示:Lva-pen 2基因是一个免疫反应相关基因,它在凡纳对虾的先天免疫系统中发挥着重要功能.     

A Toll-like receptor recognizes bacterial DNA

DNA from bacteria has stimulatory effects on mammalian immune cells, which depend on the presence of unmethylated CpG dinucleotides in the bacterial DNA. In contrast, mammalian DNA has a low frequency of CpG dinucleotides, and these are mostly methylated; therefore, mammalian DNA does not have immuno-stimulatory activity. CpG DNA induces a strong T-helper-1-like inflammatory response. Accumulating evidence has revealed the therapeutic potential of CpG DNA as adjuvants for vaccination strategies for cancer, allergy and infectious diseases. Despite its promising clinical use, the molecular mechanism by which CpG DNA activates immune cells remains unclear. Here we show that cellular response to CpG DNA is mediated by a Toll-like receptor, TLR9. TLR9-deficient (TLR9-/-) mice did not show any response to CpG DNA, including proliferation of splenocytes, inflammatory cytokine production from macrophages and maturation of dendritic cells. TLR9-/- mice showed resistance to the lethal effect of CpG DNA without any elevation of serum pro-inflammatory cytokine levels. The in vivo CpG-DNA-mediated T-helper type-1 response was also abolished in TLR9-/- mice. Thus, vertebrate immune systems appear to have evolved a specific Toll-like receptor that distinguishes bacterial DNA from self-DNA.     

基于分子对接探究熊果酸衍生物H21对引起炎症的关键蛋白的作用

利用分子对接方法,探究 H21 对分泌炎症因子的关键蛋白的影响. 从引起炎症的经典通路中选取关键靶蛋白,通过Glide分子对接,将 H21 和原配体、靶蛋白对接. 通过二者对接得到分值,筛选与 H21 结合较好的靶蛋白及关键氨基酸,并分析其二者的相互作用. 结果显示: H21 与Toll-like通路中关键蛋白IRAK1蛋白结合较好,Glide-gscore为-9.873, 优于原配体, H21 与IRAK1结合的关键氨基酸为Ile218;理论数据表明:抗炎药物 H21 可能作用于IRAK1蛋白,通过影响IRAK1蛋白的表达,从而发挥抗炎作用,为揭示 H21 的抗炎机制和作用靶点提供了理论依据.     

Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4

Signal transduction through Toll-like receptors (TLRs) originates from their intracellular Toll/interleukin-1 receptor (TIR) domain, which binds to MyD88, a common adaptor protein containing a TIR domain. Although cytokine production is completely abolished in MyD88-deficient mice, some responses to lipopolysaccharide (LPS), including the induction of interferon-inducible genes and the maturation of dendritic cells, are still observed. Another adaptor, TIRAP (also known as Mal), has been cloned as a molecule that specifically associates with TLR4 and thus may be responsible for the MyD88-independent response. Here we report that LPS-induced splenocyte proliferation and cytokine production are abolished in mice lacking TIRAP. As in MyD88-deficient mice, LPS activation of the nuclear factor NF-kappaB and mitogen-activated protein kinases is induced with delayed kinetics in TIRAP-deficient mice. Expression of interferon-inducible genes and the maturation of dendritic cells is observed in these mice; they also show defective response to TLR2 ligands, but not to stimuli that activate TLR3, TLR7 or TLR9. In contrast to previous suggestions, our results show that TIRAP is not specific to TLR4 signalling and does not participate in the MyD88-independent pathway. Instead, TIRAP has a crucial role in the MyD88-dependent signalling pathway shared by TLR2 and TLR4.     

Oncogenically active MYD88 mutations in human lymphoma

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt's lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-β. Hence, the MYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations.     

The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors

Mammalian Toll-like receptors (TLRs) function as sensors of infection and induce the activation of innate and adaptive immune responses. Upon recognizing conserved pathogen-associated molecular products, TLRs activate host defence responses through their intracellular signalling domain, the Toll/interleukin-1 receptor (TIR) domain, and the downstream adaptor protein MyD88 (refs 1-3). Although members of the TLR and the interleukin-1 (IL-1) receptor families all signal through MyD88, the signalling pathways induced by individual receptors differ. TIRAP, an adaptor protein in the TLR signalling pathway, has been identified and shown to function downstream of TLR4 (refs 4, 5). Here we report the generation of mice deficient in the Tirap gene. TIRAP-deficient mice respond normally to the TLR5, TLR7 and TLR9 ligands, as well as to IL-1 and IL-18, but have defects in cytokine production and in activation of the nuclear factor NF-kappaB and mitogen-activated protein kinases in response to lipopolysaccharide, a ligand for TLR4. In addition, TIRAP-deficient mice are also impaired in their responses to ligands for TLR2, TLR1 and TLR6. Thus, TIRAP is differentially involved in signalling by members of the TLR family and may account for specificity in the downstream signalling of individual TLRs.     

Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4

Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, and members of the pro-inflammatory interleukin-1 receptor (IL-1R) family, share homologies in their cytoplasmic domains called Toll/IL-1R/plant R gene homology (TIR) domains. Intracellular signalling mechanisms mediated by TIRs are similar, with MyD88 (refs 5-8) and TRAF6 (refs 9, 10) having critical roles. Signal transduction between MyD88 and TRAF6 is known to involve the serine-threonine kinase IL-1 receptor-associated kinase 1 (IRAK-1) and two homologous proteins, IRAK-2 (ref. 12) and IRAK-M. However, the physiological functions of the IRAK molecules remain unclear, and gene-targeting studies have shown that IRAK-1 is only partially required for IL-1R and TLR signalling. Here we show by gene-targeting that IRAK-4, an IRAK molecule closely related to the Drosophila Pelle protein, is indispensable for the responses of animals and cultured cells to IL-1 and ligands that stimulate various TLRs. IRAK-4-deficient animals are completely resistant to a lethal dose of lipopolysaccharide (LPS). In addition, animals lacking IRAK-4 are severely impaired in their responses to viral and bacterial challenges. Our results indicate that IRAK-4 has an essential role in innate immunity.     

多泛素链延伸酶E4B对TLR信号通路的调节作用

研究多泛素链延伸酶E4B对TLR信号通路的影响.在HEK293T细胞中,通过双荧光报告系统检测E4B对TLR信号通路的影响,并且用免疫共沉淀的方法鉴定E4B的相互作用蛋白质.发现多泛素链延伸酶E4B能够抑制TLR通路的下游转录因子NF-κB和IRFs的转录活性,并且能与TRAF6发生相互作用.提示多泛素链延伸酶E4B可能参与TLR信号通路的调控,为TLR信号通路的研究提供新的线索.     

广东汉族人群TLR9基因单核苷酸多态性及其单倍型(英文)

采用PCR扩增和直接测序法对191例健康且无血缘关系的广东汉族人群筛查了TLR9全基因序列,包括调控区、5′非翻译区、第1,2外显子、内含子及3′非翻译区上所有的单核苷酸多态性(SNP)位点.共检出五个SNP位点,分别为调控区的-1 486 T/C和-1 421 C/T、内含子区的+1174 A/G、第2外显子的+1 387 T/C和+2848 G/A,其中-1421 C/T和+1 387 T/C为首次发现的新位点.连锁不平衡分析表明-486 T/C,1174 A/G以及2848 G/A之间存在紧密连锁,并且涵盖了整个基因区域,形成了一个单倍域.在此基础上运用Hhase软件构建了TLR9基因的单倍型,共得到七种单倍型并模拟了它们可能的分布频率.进一步的中性检验表明TLR9基因在广东汉族人群中符合中性进化模式.