【作者】 丁彦春；

【导师】 曲鹏；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2008， 博士

【摘要】 背景和目的:血管内膜增生是动脉血管对各种损伤的一种反应,也是动脉粥样硬化和经皮冠状动脉介入治疗(percutaneous coronary intervention, PCI)术后再狭窄的重要标志。血管平滑肌细胞( vascular smooth muscle cell,VSMC)被各种损伤刺激激活后,由静息状态转变为增殖表型并移行到内膜下,参与合成各种细胞外基质,在内膜增生形成过程中具有重要作用。越来越多的证据表明感染和各种组织损伤能够通过诱导先天免疫反应,促进动脉壁的炎症,在VSMC激活和血管内膜增生过程中具有重要作用。但这些免疫反应调节和相互作用的分子生物学机制还不是很清楚。模式识别受体(pattern-recognition receptors,PRRs)是一种经过广泛选择的种系编码的受体,它们通过识别病原体关联的分子模式(pathogen-associated molecular patterns,PAMPs)来感知侵入机体的危险信号。它们与其它免疫组份一起构成了机体的第一道防线。Toll样受体(toll-like receptors,TLRs)和核苷酸结合的寡聚结构域(nucleotide- binding oligomerization domain,NOD)受体是近期确定的参与炎症疾病的两个PRRs家族。TLRs主要位于细胞表面,识别来源于细胞外部的危险信号。细菌为了避免被清除已经发展出一些侵入和通过上皮层的对策。在侵入上皮下间隙后,细菌还可以遭遇到吞噬细胞的局部抵抗,但是细菌能够利用各种对策避免被吞噬细胞的溶酶体吞食和降解而侵入细胞内。NOD受体是一种功能上与TLR相似,但位于细胞胞质溶胶的蛋白家族,它们能识别侵入细胞内的细菌产物,负责识别侵入细胞内的危险信号,启动免疫反应,代表了细胞内的一种防御监测手段。TLRs和NOD受体都具有一个富含亮氨酸的重复结构域(leucine–rich repeat domain,LRR),负责与特异的PAMPs结合。TLRs和NOD受体被激活后能启动下游衔接分子的级联反应,激活NF-κB信号传导通路,启动炎症细胞因子和其它先天免疫反应成分的转录,在清除病原感染的炎症反应中起着重要作用。NF-κB信号传导通路的激活介导了许多炎症因子的表达,这些炎症因子参与血管损伤炎症反应,在VSMC增殖、分化和凋亡的过程中起到重要的调节作用,由此推测TLRs和NOD受体介导的信号传导通路可能在血管内膜增生和VSMC激活过程中起到重要作用。本研究利用大鼠颈总动脉狭窄这一经典动物模型,观察血管内膜增生过程中TLR4/NF-κB信号传导通路的变化,以及NOD1受体在动脉损伤后增生的血管内膜中的表达和功能。比较NOD1受体在增生的内膜平滑肌细胞(iSMC)和血管中层平滑肌细胞(mSMC)中的功能。同时还对NOD1和NOD2受体在人冠状动脉VSMC中的表达及其介导的先天免疫信号传导通路在VSMC的激活过程中的作用进行研究,并探讨NOD2与TLR2、4介导的先天免疫信号传导通路在此过程中的相互关系,探讨先天免疫模式识别受体在血管内膜增生和VSMC激活过程中的作用,为防治动脉粥样硬化形成和PCI术后再狭窄提供新的思路。方法和结果:第一部分:建立大鼠颈动脉血管损伤模型,将大鼠分为正常血管组、损伤7天组和损伤14天组,用免疫组织化学染色、Western blotting和半定量反转录多聚酶联聚合反应(RT-PCR)的方法,检测了TLR4和NF-κB在损伤的大鼠颈动脉中的表达。结果发现大鼠颈动脉新生内膜在血管损伤后7天增加,增厚的内膜中以VSMC为主,损伤14天后血管内膜增生更加明显。新生的内膜平滑肌中TLR4和NF-κB染色阳性信号损伤14天组(TLR4:37.20±2.10;NF-κB:23.2±0.68)高于损伤7天组(TLR4:15.58±0.91;NF-κB:9.8±0.5),损伤7天组要高于正常组(TLR4:1.50±0.36;NF-κB:1.0±0.3)。与此一致,增生的内膜平滑肌中TLR4蛋白和mRNA表达水平也随损伤时间增加,损伤14天组(TLR4蛋白:57.1±2.2;TLR4 mRNA:0.49±0.019)高于损伤7天组(TLR4蛋白:26.2±0.9;TLR4 mRNA:0.39±0.014),损伤7天组要高于正常组(TLR4蛋白:12.0±0.5;TLR4 mRNA:0.18±0.013)。第二部分:我们用计算机比对分析的方法在大鼠基因组中确定了人类NOD1基因的类似物,并发现它们在保守的结构域上高度同源。我们应用大鼠颈动脉血管损伤模型,用免疫组织化学染色和半定量RT-PCR的方法,检测了大鼠NOD1在损伤血管中的表达。我们发现在大鼠NOD1 mRNA在颈动脉损伤后表达增加,通过免疫组化染色的方法确定了NOD1主要位于增生的内膜中。我们将增生的iSMC分离培养,并用NOD1受体特异的配体diaminopimalic acid(DAP)刺激iSMC,用实时定量RT-PCR法检测了iSMC中诱生型一氧化氮合酶(inducible nitric oxide synthase ,iNOS)和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的表达,结果发现DAP能以时间和剂量依赖的方式诱导iSMC表达iNOS。刺激NOD1还能够诱导TNF-α在iSMC中短暂的表达上调。DAP还能以时间和剂量依赖的方式诱导iSMC分泌一氧化氮(nitric oxide ,NO)。我们分别用DAP和干扰素-γ(interferon-γ,IFN-γ)单独或联合刺激iSMC和mSMC 24小时,用Western blot的方法分析NOD1、受体相互作用蛋白2(receptor-interacting protein 2,Rip2)和iNOS蛋白的表达,并检测细胞培养物上清液中NO的浓度。结果发现DAP只能诱导iSMC表达RIP2和iNOS。mSMC中没有RIP2蛋白表达。DAP和IFN-γ还能协同诱导iSMC产生NO,而在mSMC中没有发现这种协同现象。第三部分:(一)体外培养人冠状动脉VSMC,用NOD1受体的激动剂PGN(10μg/ml)刺激0、3、6和24h,用实时定量RT- PCR检测VSMC中NOD1和成纤维生长因子-2(fibroblast growth factor-2,FGF-2) mRNA的表达,用ELISA法测定细胞培养物上清夜中白细胞介素8(interleukin-8,IL-8)和TNF-α的浓度。发现VSMC在静息状态下表达低水平的NOD1。PGN刺激能够引起VSMC中NOD1 mRNA表达上调(从0.164±0.005增加到0.231±0.027,p<0.05)。伴随NOD1表达上调,PGN刺激能引起VSMC中FGF-2 mRNA快速、短暂的表达上调,在6h(22.72±0.58)达到高峰,随即下降(24h:8.85±0.96;3h:13.74±0.77; 0h:8.05±0.26)。PGN刺激能引起细胞培养物上清液中IL-8和TNF-α的分泌显著增加(IL-8从0.12±0.01 ng/ml增加到2.31±0.11ng/ml;TNF-α从4.22±0.50 pg/ml增加到143.11±12.58 pg/ml)。(二)体外培养人冠状动脉VSMC,用NOD2激动剂胞壁酰二肽(Muramyl dipeptide,MDP)、TLR2激动剂(Pam3CSK4,PAM3)和TLR4激动剂脂多糖(lipopolysaccharides,LPS)单独或联合进行刺激,用实时定量RT- PCR检测VSMC中NOD2和FGF-2 mRNA的表达,用ELISA法测定细胞培养物上清液中IL-8和TNF-α的浓度,用MTT检测VSMC增殖活性。MDP能使VSMC中NOD2 mRNA呈时间依赖性表达上调(0h:0.028±0. 001;3h:0.045±0.002;6h:0.053±0.002;24h:0.162±0.013),并能引起VSMC中FGF-2 mRNA的表达增加(9.32±0.37 vs对照组7.44±0.25;p<0.05),细胞培养物上清液中IL-8(2.41±0.22 ng/ml vs对照组1.02±0.13 ng/ml;p<0.05)和TNF-α(51.9±4.73 pg/ml vs对照组29.4±3.73 pg/ml;p<0.05)的分泌增多和细胞增殖活性增加(0.87±0.05 vs对照组0.72±0.02;p<0.05)。MDP还能协同LPS、PAM3诱导VSMC活性增加,细胞培养物上清液中IL-8和TNF-α的分泌增加。结论:1.在大鼠颈总动脉球囊损伤后再狭窄模型中,TLR4的mRNA及蛋白水平均表达增高,NF-κB活性增加,表明TLR4/NF-κB通路有可能参与了再狭窄形成的过程。2. NOD1在大鼠组织中也有表达并与人NOD1高度同源。NOD1在血管损伤修复过程中表达上调,并主要集中于增生的iSMC。iSMC中的NOD1激活后能强烈诱导iNOS和TNF-α这两个NF-　B依赖的基因的表达,表明NOD1是一个特异的血管内膜增生先天免疫反应介质。iSMC与mSMC胞浆内的NOD1受体功能的不同,这可能是由两者下游的信号通路不同所致,在mSMC中缺乏NOD1信号传导通路中关键的下游衔接分子RIP2蛋白表达。3. VSMC中NOD1介导的先天免疫信号传导通路被激活后能引起下游的促炎症因子如IL-8和TNF-α分泌增加,诱导VSMC表达FGF-2。这可能代表一个与血管炎症和内膜形成有关的新的先天免疫反应激活通路。4. NOD2介导的先天免疫信号传导通路的激活能导致VSMC增殖活性增加,诱导其分泌促炎症细胞因子。该信号通路与TLR2、4介导的先天免疫信号传导通路在促进VSMC增殖活性增加,诱导VSMC分泌促炎症细胞因子的过程中具有协同作用。更多还原

【Abstract】 Background and Objective: Vascular intimal hyperplasia is a response of the arterial wall upon a wide range of stimuli. It is also a prominent hallmark of atherosclerosis and restenosis after percutaneous coronary intervention (PCI). Activation of vascular smooth muscle cells (VSMC), which switch from quiescent state to proliferative phenotype, migrates into the intima to proliferate and produce extracellular matrix proteins, is a key event in intimal hyperplasia in response to inflammation or mechanical injury. More and more evidences show that infections and injurious stimuli play an important role in the activation of VSMC and intimal hyperplasia by triggering innate immune responses and promoting inflammation in the vessel wall. Yet, underlying molecular bases remain undefined.Pattern recognition receptors (PRRs) represent a diverse collection of germline-encoded receptors responsible for detecting danger signals by sensing pathogen-associated molecular patterns (PAMPs), and together with other immune components they are involved in the first line of defence. Two families of PRRs, toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) receptors have recently been identified and implicated in inflammatory diseases. Among them, TLRs are expressed on the surface of cells and responsible for detecting extracelluar PAMPs. To avoid recognition and escape from defense, bacteria have developed strategies for invading and crossing the epithelium. Upon arrival at the subepithelial space, bacteria encounter locally resident, as well as newly infiltrated, professional phagocytic cells. However, bacteria use a variety of strategies to avoid engulfment and degradation by the endolysosomal compartment by phagocytes. NOD receptors have the similar function of TLRs, which are cytoplasmic proteins, involved in intracellular bacterial detection, representing a means of cytosolic surveillance.Both TLRs and NOD receptors have a leucine rich repeat domain, which is involved in specific PAMPs recognition. Activation of TLRs and NOD receptors can initiate downstream cascade reaction of adapter molecules, which eventually lead the activation of nuclear factor-κB (NF-κB) and initiate the transcription of inflammatory cytokines and other host response elements, play a key role in the clearance of infecting pathogens. NF-κB signal transduction pathway play a central role in regulating VSMC proliferation, differentiation and apoptosis by inducing the expression of many kinds of proinflammatory cytokines. Quibus we suppose the signal pathway mediated by TLR or NOD receptors may play an important role in the VSMC activation and intimal hyperplasia.To explore the role of PRRs in the VSMC activation and intimal hyperplasia, and provide a novel insight for preventing atherosclerosis and restenosis, we explore the expression of TLR4/NF-κB and NOD1 signal pathway during the progression of intimal hyperplasia by a well established animal model of restenosis of rat balloon injury of the common carotid artery. We also observe the expression and function of NOD1 in the rat intimal smooth muscle cells (iSMC), and the NOD1 functions difference in iSMC and medial SMC (mSMC). We further investigate the expression of NOD1 and NOD2 in human VSMC, and the role of NOD1 and NOD2 mediated innate immune signaling pathway in the activation VSMC. We also explore the possible interaction of NOD2 signal pathways with TLR2 and 4 signal pathways in the induction of proinflammatory cytokines in VSMC.Methods and Results: Part one: We use immunohistochemistry, Western blotting and semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) to detect the expression of TLR4 and NF-κB on rat carotid arteries following angioplastic injury. We found the level of TLR4 both in mRNA and protein was increase following the neointimal formation. The expression of NF-κB was also up-regulated with intima hyperplasia.Part two: Using the methods of immunohistochemistry and semiquantitative RT-PCR, we found that expression of human homologue of NOD1 gene was highly up-regulated with the development of intima after angioplastic injury to rat carotid artery, and predominantly localized in intimal lesion. Studies using isolated iSMC showed that stimulation of NOD1 by its ligand diaminopimalic acid (DAP) led to marked induction of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in time-depend and dose-depend manner. Stimulation of NOD1 also led to a transient up-regulation of tumor necrosis factor-α?TNF-α??in iSMC. Rat iSMCs and mSMC were stimulated with DAP and/or IFN- ?for 24 hours. Expression of NOD1 and receptor-interacting protein 2(RIP2) and iNOS were assessed by Western blot analysis. In spite of comparable levels of NOD1 protein in iSMCs and mSMC under basal condition, activation of RIP2 and expression of iNOS were elicited merely in iSMC subsequent to the stimulation. In addition, a synergistic effect of DAP and IFN- ?was shown in the induction of NO production by iSMC whereas such effect could not be found in mSMC.Part three: (1) Human coronary artery SMCs were in vitro stimulated with NOD1 receptor agonist PGN(10μg/ml)for 0, 3, 6 and 24 hours. The mRNA expression of NOD1 and fibroblast growth factor-2(FGF-2) were measured by real time quantitative RT-PCR. The concentration in the culture supernatants of interleukin-8(IL-8) and TNF-αwas determined by ELISA. We found human SMC constitutively expressed a low level of NOD1 under resting condition. Upon PGN stimulation, the expression of NOD1 mRNA was up-regulated in SMC, from 0.164±0.005 to 0.231±0.027 (p< 0.05). Stimulation of NOD1 also led to a transient up-regulation of FGF-2, which reaches the peak at 6 hour(22.72±0.58), then decrease quickly(24h:8.85±0.96;3h:13.74±0.77; 0h:8.05±0.26)。ELISA showed the concentration in the culture supernatants of IL-8 and TNF-αincreased from 0.12±0.01ng/ml to 2.31±0.11 ng/ml and from 4.22±0.50 pg/ml to 143.11±12.58 pg/ml, respectively (p< 0.01).(2) Human coronary artery SMCs were in vitro stimulated with NOD2 agonist Muramyl dipeptide (MDP), TLR2 agonist Pam3CSK4(PAM3)and TLR4 agonist lipopolysaccharides (LPS) alone or MDP in synergy with either PAM3 or LPS. The mRNA expression of NOD2 and FGF-2 were measured by real time quantitative RT-PCR. The concentration in the culture supernatants of IL-8 and TNF-αwas determined by ELISA. VSMC proliferation viability was analyzed by the MTT assay. MDP can up-regulate the expression of NOD2 mRNA as a time-dependent manner (0h: 0.028±0. 001; 3h: 0.045±0.002; 6h: 0.053±0.002; 24h: 0.162±0.013) in VSMC. It can also up-regulate the expression of FGF-2 mRNA (9.32±0.37 vs control 7.44±0.25; p<0.05) in VSMC, induce the production of IL-8 and TNF-α(2.41±0.22 ng/ml vs control 1.02±0.13 ng/ml; 51.9±4.73 pg/ml vs control 29.4±3.73 pg/ml; respectively, p < 0.05) and increase the proliferation viability of VSMC (0.87±0.05 vs control 0.72±0.02; p<0.05). Additional, MDP can synergy with LPS and PAM3 to increase the proliferation viability of VSMC and induce the production of IL-8 and TNF-α.Conclusion: 1.In the model of vascular restenosis by balloon injury, the levels of TLR4 mRNA and protein of carotid arteries were significantly increased and companied with activation of NF-κB. It implied that TLR4/ NF-κB pathway may play an important role in the developing of restenosis. 2. NOD1 is also expressed in rat tissue and exhibits highly homology with the human NOD1. Rat NOD1 has an increased expression during intimal hyperplasia in a rat carotid artery injury model and preferentially expressed by iSMCs. Activation of NOD1 can robustly induce the expression of iNOS and TNF-α, which regulated by NF-κB signal pathway. It implied that NOD1 is an intimal specific innate immune response mediator. The functional difference of NOD1 in iSMC and mSMC maybe due to the difference of their downstream mediator. The mSMC lacks RIP2 protein, a key component of NOD1 signal pathway.3. The activation of NOD1 mediated innate immune signaling pathway can induce the production of proinflammatory cytokines such as IL-8 and TNF-αin VSMC. It can also induce the expression of FGF-2. This may represent a novel innate immune activation pathway in vascular inflammation and intimal formation.4. The activation of NOD2 mediated innate immune signaling pathway can increase the proliferation viability of VSMC and induce the production of proinflammatory cytokines in VSMC. It had a synergistic effect with those of TLR2 and TLR4 mediated signaling pathways in this process.更多还原