【作者】 胡震；

【导师】 丁素菊； 曲乐丰；

【作者基本信息】 第二军医大学 ， 神经病学， 2012， 博士

【摘要】 炎症反应在动脉粥样硬化的发病和进展过程中起关键作用，其中感染引起的炎症反应与动脉粥样硬化的关系日益受到重视。最新的研究认为幽门螺杆菌（Helicobacterpylori，Hp）、肺炎衣原体、巨细胞病毒、单纯疱疹病毒等病原微生物感染是动脉粥样硬化的重要致病因素之一，其中Hp感染与动脉粥样硬化性疾病的相关性研究是近年来的热点。但是，目前国内外的研究多局限于Hp促动脉粥样硬化现象的观察和描述，缺少相关机制研究，Hp是如何促进动脉粥样硬化发展的机制尚不清楚。Hp属革兰阴性杆菌，脂多糖（LPS）是革兰阴性菌的重要致病因素。根据“动脉粥样硬化即炎性病变”的理论，Hp的感染及其LPS所诱发的炎症和免疫反应在动脉粥样硬化的发展中可能起到更重要的作用。本研究分为以下几个方面：（1）人体水平：对动脉粥样硬化患者行颈动脉彩超观察斑块大小、性质及颈动脉内膜中层厚度（CIMT），收集颈动脉粥样硬化患者血清行Hp抗体及与动脉粥样硬化稳定性相关的炎症因子检测；（2）病理水平：对有手术指针患者行颈动脉内膜剥脱术（CEA），取得粥样硬化斑块切片免疫组化染色观察Hp感染患者粥样硬化斑块结构及炎症因子表达的变化；（3）细胞水平：通过Hp LPS干预RAW264.7巨噬细胞，观察Hp LPS对巨噬细胞吞噬氧化低密度脂蛋白的影响和释放炎症因子的变化；（4）膜受体水平：通过基因敲除动物，明确Hp LPS对巨噬细胞造成影响相关的Toll样受体类型。通过本研究明确幽门螺杆菌感染对颈动脉粥样硬化斑块稳定性的影响及Hp LPS在促进动脉粥样硬化中的作用机制，为动脉粥样硬化的发病机制提供新的视角，为脑卒中的预防提供新的理论依据和防治策略。第一部分幽门螺杆菌感染对颈动脉粥样硬化超声形态及血清炎症因子的影响目的：在本实验通过检测动脉粥样硬化患者颈动脉彩超和血清MPO、MMP-9、LP-PLA2，观察幽门螺杆菌感染对动脉粥样硬化斑块形态、CIMT及血清炎症因子的影响。分析Hp感染对动脉粥样硬化斑块稳定性的影响，探讨其感染导致颈动脉粥样硬化形成的可能机制方法：选择2011年4月到2011年9月在我院行颈动脉彩超提示动脉粥样硬化的知情同意者141例，其中男患者85例，女患者56例。行血清幽门螺杆菌抗体（Hp IgG）检测，根据患者血清幽门螺杆菌抗体结果分为幽门螺杆菌阳性组和幽门螺杆菌阴性组。进行血清幽门螺杆菌抗体（Hp-IgG）检测，颈总动脉内膜中层厚度检测，颈动脉粥样斑块形态的检测和血清MPO、MMP-9、LP-PLA2的检测。结果：1. Hp阳性组颈动脉不稳定斑块29个，其中软斑24个，溃疡斑块5个；稳定斑块43个，其中扁平斑块26个，硬斑17个。Hp阴性组颈动脉不稳定斑块17个，其中软斑14个，溃疡斑块3个；稳定斑块52个，其中扁平斑块38个，硬斑14个。同Hp阴性组相比，Hp阳性组患者的不稳定性斑块发生率增高，并有统计学差异（P=0.0477）。2.通过颈动脉超声观察各组患者双侧颈总动脉CIMT, Hp阳性组左侧颈总动脉CIMT为0.096±0.0013，Hp阴性组左侧颈动脉CIMT为0.087±0.0019，阳性组较阴性组的左侧颈总动脉CIMT有显著增厚（P=0.0003）。Hp阳性组右侧颈总动脉CIMT为0.093±0.0011，Hp阴性组右侧颈动脉CIMT为0.086±0.0021，阳性组较阴性组的右侧颈总动脉CIMT有显著增厚（P=0.0041）。3. Hp阳性组血清MPO浓度为767.6±11.06ng/ml，较Hp阴性组707.2±15.38ng/ml显著增加（P=0.0017）。Hp阳性组血清hs-CRP浓度为4.257±1.102mg/dl，较Hp阴性组4.408±1.174mg/dl无显著差异。（P=0.0925）。Hp阳性组血清MPO浓度为21.40±0.2639ng/ml，较Hp阴性组19.80±0.4886ng/ml显著增加（P=0.0041）。Hp阳性组血清LP-PLA2浓度为393.8±4.855ng/ml，较Hp阴性组366.7±9.113ng/ml显著增加（P=0.0090）。结论：1.本研究通过临床证实，Hp感染可引起动脉粥样硬化斑块不稳定性增加。2.Hp感染的动脉粥样硬化患者血清MPO、MMP-9、LP-PLA2增高，而hs-CRP增高不显著。第二部分幽门螺杆菌感染者颈动脉粥样硬化斑块结构分析及炎症因子表达的变化目的：为了明确幽门螺杆菌感染者颈动脉粥样硬化斑块内炎症因子表达的变化，本实验通过对有手术指针的患者行CEA手术获得颈动脉粥样硬化斑块组织进行病理检测，行HE及免疫组化染色，观察其形态及炎症因子的表达与幽门螺杆菌感染的关系。方法：从第一部分所研究的141例对象中，选择符合行颈动脉内膜切除术（carotidendarterectomy, CEA）的36例患者行CEA手术。Real-Time PCR检测颈动脉粥样斑块组织内炎症因子的mRNA变化，免疫组化检测颈动脉粥样硬化斑块组织的炎症因子表达。结果：1. Real-time PCR结果显示Hp阳性组MPO的mRNA的相对GAPDH的表达量为3.096±0.2846，较Hp阴性组1.924±0.2535显著升高（P=0.0051）；Hp阳性组MMP-9的mRNA的相对GAPDH的表达量为2.212±0.3147，较Hp阴性组1.406±0.1907显著升高（P=0.0472）；Hp阳性组LP-PLA2的mRNA的相对GAPDH的表达量为7.013±0.9494，较Hp阴性组3.614±0.8607显著升高。2、Hp阳性组纤维帽厚度92.10±11.26μm，Hp阴性组纤维帽厚度130.9±12.43μm，Hp阴性组较阳性组纤维帽厚度明显增厚（P=0.0269）。Hp阳性组脂质池面积比为0.6172±0.03054， Hp阴性组脂质池面积比为0.5324±0.03702，Hp阳性组较阴性组脂质池面积增加，但差异不显著（P=0.0836）。3、同Hp阴性组相比较Hp阳性组动脉粥样硬化板块中的LP-PLA2、MPO、MMP-9表达均有明显增高。结论：1.进一步证实，同不伴有Hp感染者相比，Hp感染者患颈动脉粥样硬化的危险性更大。2.Hp感染可能通过增强粥样硬化斑块局部的炎症反应及免疫反应促进和加重颈动脉粥样硬化形成。3. Hp感染者颈动脉粥样硬化局部炎症因子释放增多，从而影响颈动脉粥样硬化的形成。第三部分幽门螺杆菌脂多糖对巨噬细胞的影响目的：因此为了明确幽门螺杆菌的在促动脉粥样硬化中的作用机制，本研究利用提纯的幽门螺杆菌脂多糖干预体外培养的巨噬细胞，观察巨噬细胞吞噬氧化低密度脂蛋白的变化以及释放的炎症因子改变。方法提取和纯化幽门螺杆菌脂多糖，干扰培养的RAW264.7单核巨噬细胞，通过荧光探针Dil、油红O染色、酶荧光法定量观察巨噬细胞吞噬ox-LDL能力变化，并检测IL-6、TNF-α的mRNA观察巨噬细胞炎症因子释放情况，同时检测TLR2、TLR4的mRNA观察巨噬细胞受体的改变。结果：1、可见加入Hp LPS组的巨噬细胞荧光明显增加，表明其吞噬的ox-LDL增加， ox-LDL+Hp LPS组巨噬细胞内胆固醇脂（CE）与较ox-LDL组明显增加（P=0.0023）。2、Real-time PCR结果显示不同时间Hp LPS刺激后，巨噬细胞的IL-6及TNF-α的mRNA变化趋势，其中以2-4小时变化最为显著(P<0.05)。3、当给予不同梯度稀释的Hp LPS刺激巨噬细胞后，其TLR2受体mRNA水平有上升趋势（P<0.01），而TLR4的mRNA水平则上升趋势不显著（P>0.05）。结论：本研究通过提纯幽门螺杆菌脂多糖，并用其干预体外培养的RAW264.7单核巨噬细胞，发现巨噬细胞吞噬ox-LDL增加，促进了巨噬细胞向泡沫细胞转变，并引起巨噬细胞释放IL-1β、IL-6、TNF-α和MMP-9的增加。同时还观察到幽门螺杆菌脂多糖可促进巨噬细胞表面TLR2受体的表达增加，表明幽门螺杆菌脂多糖可能通过TLR2促进巨噬细胞向泡沫细胞转变及释放炎症介质。第四部分TLR2基因敲除对Hp LPS促巨噬细胞泡沫化的影响目的：通过Hp LPS干预TLR2基因敲除小鼠和C57BL/6J小鼠的腹腔巨噬细胞，检测其吞噬ox-LDL的变化以及TLR2、TLR4、胆固醇代谢受体和炎症因子的表达变化，明确Hp LPS作用于巨噬细胞的受体及探讨其促进巨噬细胞泡沫化的机制。方法：分别取C57BL/6J小鼠和TLR2基因敲除小鼠的腹腔巨噬细胞，给予（Dil-）ox-LDL、（Dil-）ox-LDL+Hp LPS、（Dil-）ox-LDL、（Dil-）ox-LDL+Hp LPS处理。通过荧光探针Dil、酶荧光法定量观察巨噬细胞吞噬ox-LDL能力变化。并检测巨噬细胞IL-6、TNF-α的mRNA变化和巨噬细胞TLR受体及胆固醇代谢受体检测巨噬细胞CD36和ABCG1的mRNA变化。结果：1、从荧光显像可见TLR2两组细胞荧光密度均较C57组降低，说明TLR2-/-小鼠腹腔巨噬细胞吞噬氧化低密度脂蛋白能力较C57小鼠吞噬能力降低。2、结果显示ox-Hp LPS组巨噬细胞内胆固醇脂（CE）与较ox-LDL组明显增加（P=0.0023）。3、Real-time PCR结果显示C57小鼠巨噬细胞受Hp LPS刺激后IL-6及TNF-α的mRNA变化较TLR2敲除小鼠明显增加。4、结果显示Hp LPS刺激后ABCG1的mRNA下降，而CD36的mRNA水平上调。其中LPS+LDL组与LDL组比较有显著差异（P<0.05）。结论：1、Hp LPS可通过TLR2促进巨噬细胞吞噬ox-LDL，并促进其释放炎症介质2、Hp LPS可通过TLR2可促进ABCG1的mRNA下降，而CD36的mRNA水平上调。3、Hp LPS可能激活了PPARγ通路促进CD36的高表达，使巨噬细胞吞噬氧化低密度脂蛋白能力增加，同时抑制了LXRα通路，导致ABCG1低表达，使巨噬细胞胆固醇流出减少，两者协同作用，促进了动脉粥样硬化的发展。更多还原

【Abstract】 The inflammatory response plays a key role in the incidence and progression ofatherosclerosis, in which infection-induced inflammation and atherosclerosis relationshiphave received increasing attention. The latest studies suggest that Helicobacter pylori (Hp),Chlamydia pneumoniae, cytomegalovirus, herpes simplex virus infection of pathogenicmicroorganisms is one of the important risk factors for atherosclerosis, in which Hpinfection and atherosclerosis sclerosing disease is a hot topic in recent years. However,studies at domestic and international was restricted to observation and description of thephenomena that Hp promote atherosclerosis, lacking of mechanisms study. How Hppromote the development of atherosclerosis mechanism is still unclear. Hp is a type ofGram-negative bacteria, lipopolysaccharide (LPS) is an important pathogenic factor ofGram-negative bacteria. According the theory "atherosclerosis is an inflammatory disease"we conclude that Hp infection and LPS-induced inflammation and immune response mayplay a more important role in the development of atherosclerosis.The study is divided into the following aspects:(1)The body level: ultrasonographyobserve the size, nature and the carotid artery intima-media thickness (CIMT) of plaque incarotid atherosclerosis patients. Collect serum of carotid atherosclerosis patients and detectof Hp antibody and inflammatory factors related to the stability of atherosclerotic;(2) Thepathology level: obtain the atherosclerotic plaques by carotid endarterectomy surgery(CEA). Observe the changes in atherosclerotic plaque structure and expression ofinflammatory cytokines by immunohistochemical stained;(3) The cellular level: StimulateRAW264.7macrophages with Hp LPS, and observe the change of macrophagephagocytosis of oxidized low-density lipoprotein and the release of inflammatory factorsunder the influence of Hp LPS;(4) membrane receptor level: Through the knockoutanimals, clarify the type of Toll-like receptor on macrophages stimulated by Hp LPS. Thisstudy clarified the impact of Helicobacter pylori infection to carotid atherosclerotic plaquestability and the mechanism of Hp LPS promoting atherosclerosis. The stydy not onlyoffered new perspective for pathogenesis of atherosclerosis, but also offered newtheoretical basis and control strategies for stroke prevention. PART Ⅰ The Impact of Helicobacter Pylori Infection on UltrasoundMorphology of Carotid Atherosclerosis and SerumInflammatory CytokinesObjective: In this experiment, we observed the impact of H. pylori infection onarterial atherosclerotic plaque morphology, CIMT and serum inflammatory cytokines, bydetecting arterial atherosclerosis in patients with carotid ultrasound and serum MPO,MMP-9, LP-PLA2dencity, Analysis of Hp infection on atherosclerotic plaque stability,and to explore its possible mechanisms of infection lead to carotid artery atherosclerosisMethods:141cases of carotid artery ultrasonography in our hospital from April2011to September2011prompted atherosclerosis informed consent,85cases were malepatients,56cases of female patients. Line serum Helicobacter pylori (Hp IgG) weredetected in patients with Helicobacter pylori antibody results into the H. pylori-positivegroup and the H. pylori-negative group. Serum Helicobacter pylori antibodies (Hp-IgG)testing, carotid artery intima-media thickness detection, detection of carotid artery plaquemorphology and serum of MPO and MMP-9, LP-PLA2detected.Results:1.29unstable carotid plaque in Hp-positive group, including24soft plaquesand5ulcer plaques;43stable plaques, including flat plaques26and17hard plaques.17unstable carotid plaques in Hp-negative group, including14soft plaques and3ulcersplaques;52stable plaques, including38flat plaques and14hard plaques. WithHp-negative group compared to the Hp positive group of patients with unstable plaqueincidence increased, and there is a significant difference (P=0.0477).2. Observed throughthe carotid artery ultrasound Hp-positive group of patients in each group of bilateralcommon carotid artery CIMT values of the left common carotid artery CIMT value of0.096±0.0013, Hp-negative group, the left common carotid artery CIMT value of0.087±0.0019, positive group than in the negative The left common carotid artery CIMT groupsignificantly thicker (P=0.0003). Hp-positive group right common carotid artery CIMTvalue of0.093±0.0011, Hp-negative group, the right common carotid artery CIMT valueof0.086±0.0021, the positive group than negative group, the right common carotid arteryCIMT was significantly thickened (P=0.0041).3.Serum MPO concentration ofHp-positive group was767.6±11.06ng/ml compared to Hp-negative group,707.2±15.38ng/ml a significant increase (P=0.0017). Serum hs-CRP concentrations ofHp-positive group was4.257±1.102mg/dL, compared with Hp-negative group,4.408± 1.174mg/dl no significant difference.(P=0.0925). Hp-positive serum MPO concentrationof21.40±.2639ng/ml compared to Hp-negative group,19.80±0.4886ng/ml asignificant increase (P=0.0041). Hp-positive serum LP-of PLA2concentration was393.8±4.855ng/ml compared to Hp-negative group366.7±9.113ng/ml, a significantincrease (P=0.0090).Conclusion:1. In this study, confirmed by clinical, atherosclerotic plaque instability beincreased caused by Hp infection.2. Artery atherosclerosis patients serum MPO, MMP-9and LP-PLA2but not hs-CRP increased with Hp infection.PART Ⅱ Helicobacter Pylori infection and Carotid AtheroscleroticPlaque Results Changes of Structure and Expression ofInflammatory CytokinesObjective: In order to clarify Helicobacter pylori infection and carotid atheroscleroticplaque inflammation factors expression changes，this experiment treated surgery pointerpatients with CEA surgery.And we made pathological examination in atheroscleroticplaque tissue, by HE and immunohistochemical staining, to observe the morphology andthe expression of inflammatory factors.Methods:141cases from the first part of the research object,36patients of witchunderwent CEA surgery. Real-Time PCR detection of carotid atherosclerotic plaque withinthe organization of inflammatory factors mRNA, Immunohistochemical detection ofcarotid atherosclerotic plaques tissues, expression of inflammatory cytokines.Results:1.The Real-time PCR results showed that Hp-positive group of MPO mRNArelative GAPDH expression level was3.096±0.2846, significantly higher compared toHp-negative group,1.924±0.2535(P=0.0051); Hp positive MMP-9mRNA relativeGAPDH expression level was2.212±0.3147, significantly higher than Hp-negative group1.406±0.1907(P=0.0472); Hp positive group LP-PLA2mRNA relative GAPDHexpression level was7.013±0.9494, were significantly increased compared to Hp-negativegroup (3.614±0.8607).2., fibrous cap thickness of the Hp positive group was92.10±11.26μm, fibrous cap thickness of the Hp-negative group was130.9±12.43μm. Hpnegative group’s fibrous cap thickness was significantly thicker than the positive group (P=0.0269). lipid pool area proportion of Hp positive group was0.6172±0.03054, lipidpool area proportion of the Hp negative group was0.5324±0.03702. lipid pool area increased in Hp-positive group, but the difference was not significant (P=0.0836).3.Hp-positive group of atherosclerotic plates in the LP-of PLA2, MPO and MMP-9expression were significantly increased compared with the Hp negative group.Conclusions:1. Further confirmed, compared Hp negative group, Hp-infected personswere suffering from a greater risk of carotid atherosclerosis.2.Hp infection may enhancingthe atherosclerotic plaque inflammatory response and immune response and increasedcarotid artery atherosclerosis.3.Hp infected with carotid artery atherosclerosis localinflammatory factors increased, thus promoting the formation of carotid atherosclerosis.PART Ⅲ Affection to Macrophages by Helicobacter PyloriLipopolysaccharideObjective: In order to clarify the mechanism of H. pylori infection to promoteatherosclerosis, we intervented vitro cultured macrophages with purified H. pylorilipopolysaccharide. Observed macrophage phagocytosis of oxidized low-densitylipoprotein and release of inflammatory cytokines.Methods: Interference cultured RAW264.7monocyte-macrophage cells with purifedHelicobacter pylori lipopolysaccharide. Observed tha change of macrophage phagocytosisox-LDL ability by fluorescent probes Dil, oil red O staining, enzyme fluorescent. Observerelease of macrophage inflammatory factor by detected IL-6, TNF-alpha mRNA, and at thesame time,we detect TLR2and TLR4mRNA to observe macrophage receptors.Results:1.macrophage fluorescence was significantly increased stimulated by Hp LPS,indicating that the increase of the phagocytosis of ox-LDL. And the macrophagecholesteryl ester (CE) in ox-LDL+Hp LPS group was significantly increased comparedwith ox-LDL group (P=0.0023).2.Real-time PCR results showed that at different times ofHp LPS stimulation of macrophage IL-6and TNF-alpha mRNA trends, the mostsignificant change was2-4hours (P <0.05).3. When macrophages was stimulated bydifferent dilutions of Hp LPS, TLR2receptor mRNA levels is an upward trend (P <0.01),and TLR4mRNA level rise trend was not significant (P>0.05).Conclusion: This study intervent vitro RAW264.7monocyte-macrophage cells bypurified H. pylori lipopolysaccharide.We found out that macrophage phagocytosis ofox-LDL increased, promoting the transformation of macrophages into foam cells, and cause macrophages release IL-6, TNF-alpha increased. Also observed that Helicobacterpylori lipopolysaccharide can promote the increase expression of TLR2receptors onmacrophage cell surface, indicating that H. pylori lipopolysaccharide may promote thetransformation of macrophages to foam cells and release of inflammatory mediatorsthrough TLR2.Part Ⅳ Hp LPS Promote Macrophage Foam Cells Affected by TLR2Gene KnockoutObjective: Intervent peritoneal macrophages of the TLR2gene knock out mice andC57BL/6J mouse by Hp LPS,and detect changes in phagocytosis ox-LDL and TLR2/TLR4,cholesterol metabolism, receptors and inflammatory cytokines expression. Clarify Hp LPSeffect on macrophage receptors and to explore the mechanism of its catalytic macrophagefoam cells.Methods: Lavage peritoneal macrophages from C57BL/6J and TLR2gene knock outmouse, dealed with（Dil-）ox-LDL、（Dil-）ox-LDL+Hp LPS、（Dil-）ox-LDL、（Dil-）ox-LDL+Hp LPS. Observed macrophage phagocytosis of ox-LDL ability by fluorescentprobe Dil, the statutory amount of the enzyme fluorescence. And detect IL-6, TNF-alphaand TLR receptors mRNA of macrophages and cholesterol metabolism in receptordetection macrophage CD36and of ABCG1mRNA.Results:1.It can be seen from the fluorescence imaging that fluorescence intensitydecreased in TLR2-/-cells compared with the C57group, indicating that TLR2-/-mouseperitoneal macrophage phagocytic activity lower than C57mice.2.The results showed thatcholesteryl ester (CE) in macrophages of ox-Hp LPS group increased significantly thanox-LDL group.(P=0.0023).3. Real-time PCR results showed that the IL-6and TNF-alphamRNA of C57mice were significantly increased than the TLR2knockout mice stimulatedby Hp LPS.4. Real-time PCR results showed that ABCG1mRNA decreased, and CD36mRNA levels increase stimulated by Hp LPS, in which LPS+LDL group weresignificantly different with the LDL group (P <0.05).Conclusion:1. Hp LPS can promote macrophage phagocytosis of ox-LDL, andrelease inflammatory mediators through TLR2.2. Hp LPS can promote ABCG1mRNAdecreased, with CD36mRNA levels increased through TLR.3. Hp LPS may activate PPARγ pathway to promote high expression of CD36, increased macrophage phagocytosisof oxidized LDL capacity, while inhibiting LXRα pathway, cause ABCG1low expression,and macrophage cholesterol efflux to reduce both coordination role in promoting thedevelopment of atherosclerosis.更多还原