【作者】 张冬梅；

【导师】 潘亚萍；

【作者基本信息】 中国医科大学 ， 口腔临床医学， 2008， 博士

【摘要】 牙周炎是一种由菌斑细菌引发的慢性感染性疾病,是人类口腔两大疾病之一,发病率较高。大量的研究显示慢性牙周炎已成为心血管疾病的独立危险因素,使动脉粥样硬化(atherosclerosis, AS)和冠心病等心血管疾病的患病风险大大增加。以往的理论认为AS主要是局部脂质堆积性疾病,近年来,AS发病机制的炎症观点又重新被强调。目前研究认为AS是一个进行性的炎症反应,局部和远隔部位感染可以促进AS的慢性炎症过程。由于AS病因复杂,既包括与脂蛋白代谢有关的基因,也包括环境因素的影响,且病变发生较慢,在早期无症状,故对其病因和发病机制的研究进展仍然较慢。而大多数的牙周疾病是可以预防和控制的,有效的牙周治疗、积极的控制口腔感染为我们提供了一条减少心血管疾病发生危险的可能途径。牙龈卟啉单胞菌(Porphyromonas gingivalis, P. gingivalis)是目前公认的慢性牙周炎重要可疑致病菌,它的多种毒性因子可引发宿主的免疫炎症反应,导致牙龈组织破坏和局部血管改变,使菌血症发生的可能性和严重性大大增加。Haraszthy和Kozarov等用PCR的方法证实AS斑块内P. gingivalis的存在；Lalla和Gibson等的动物实验研究显示P. gingivalis通过口腔感染载脂蛋白E缺陷小鼠可增加早期AS和血管炎症的发生。还有研究表明P. gingivalis能够进入血液循环系统,进而粘附、侵入血管内皮细胞,并在细胞内增殖,这使P. gingivalis感染与AS疾病的联系更加密切。同其他血管壁细胞一样,血管内皮细胞可能作为P. gingivalis菌体或菌体成分的储存库,在P. gingivalis感染机体的过程中刺激机体产生免疫炎症反应。在AS疾病发生过程中,血管内皮细胞是单核细胞粘附聚集的关键部位,细胞-细胞、细胞-基质间相互粘附、相互作用几乎贯穿了AS的整个病理过程,因此了解引起细胞间相互作用的因素及机理,采取相应的措施,对防治血管病发生、发展有着重要意义。细胞间粘附的分子基础是粘附分子表达,在体内正常细胞不表达或仅轻微表达粘附分子,在AS斑块部位发现内皮细胞活化,大量表达细胞粘附分子,包括细胞间粘附分子1(intercellular adhesion molecule 1, ICAM-1)、血管细胞粘附分子1(vascular cell adhesion molecule 1, VCAM-1)和E选择素等,其作用主要是通过识别并结合对方细胞膜上的特异性配体而完成细胞间的粘附。内皮细胞分泌的粘附分子对AS病变的形成和发展有重要作用。我们推测P. gingivalis侵入血管内皮细胞可导致粘附分子高表达的AS样改变。本研究建立P. gingivalis侵入血管内皮细胞模型,模拟P. gingivalis感染血管壁细胞的体内环境,观察P. gingivalis侵入后粘附分子ICAM-1、VCAM-1和E选择素表达的变化,探讨P. gingivalis在AS疾病发生中的可能作用,并进一步完成调控细胞粘附分子表达的信号通路的初探。材料与方法一、实验材料P. gingivalis低毒力株ATCC 33277(由首都医科大学口腔医学院科研所提供)和高毒力株W83(由美国佛罗里达大学牙科学院口腔生物研究所Dr. Lamont RJ教授惠赠)，人脐静脉血管内皮细胞(human umbilical vain endothelial cell, HUVEC)株ECV304(购自南京凯基生物科技开发有限公司),甲基噻唑基四唑(methyl thiazolyl tetrazolium, MTT) (Sigma), Annexin-V-FITC凋亡检测试剂盒(深圳晶美生物技术有限公司),碘化丙啶(propidium iodide, PI) (Sigma),DNA抽提试剂盒(Qiagen),RNA提取试剂Trizol Reagent (Invitrogen), RT-PCR试剂盒(TaKaRa),鼠抗人ICAM-1单克隆抗体(Santa Cruz),兔抗人VCAM-1、E选择素多克隆抗体(Santa Cruz),兔β-肌动蛋白(actin)多克隆抗体(北京博奥森生物技术有限公司),鼠抗人IκB-α、磷酸化p38MAPK、NF-κB p65单克隆抗体(Santa Cruz),碱性磷酸酶标记的羊抗鼠或羊抗兔IgG(北京博奥森生物技术有限公司),异硫氰酸荧光素(Fluorescein isothiocynate, FITC)标记的羊抗鼠或羊抗兔IgG(北京博奥森生物技术有限公司),MG132 (CALIOCHEM), SB203580 (Promega)二、实验方法1、选择P. gingivalis低毒力株ATCC 33277和高毒力株W83、人脐静脉血管内皮细胞株,建立P. gingivalis ATCC 33277和W83侵入HUVEC模型。2、MTT比色法检测P. gingivalis侵入前后细胞增殖活性变化,流式细胞仪测定细胞周期。3、Annexin V-FITC凋亡试剂盒检测细胞凋亡率,免疫荧光染色和细胞核DNA阶梯状图谱电泳分析进一步证实细胞凋亡4、RT-PCR和Western印迹法检测P. gingivalis侵入血管内皮细胞前后ICAM-1、VCAM-1和E选择素基因的mRNA及蛋白表达变化,细胞免疫荧光染色法测定ICAM-1、VCAM-1和E选择素膜蛋白表达。5、Western印迹法检测IκB-α和磷酸化p38 MAPK表达；间接免疫荧光法检测HUVEC NF-κB p65核移位。6、NF-κB抑制剂MG132和p38 MAPK抑制剂SB203580预处理细胞后,分别建立P. gingivalis ATCC 33277和W83侵入模型,分析NF-κB/IκB和p38 MAPK信号通路在P. gingivalis侵入血管内皮细胞影响粘附分子表达过程中的作用。三、统计分析结果以均数±标准差(x±s)表示,实验重复3次,采用SAS 8.12软件包,多组均数之间的比较采用重复测量资料的方差分析,P<0.05为差异有统计学意义。实验结果1、牙龈卟啉单胞菌侵入对血管内皮细胞增殖和凋亡的影响P. gingivalis侵入后48h,未见细胞增殖受抑制。与对照组比较,P. gingivalis ATCC 33277侵入后72h,细胞增殖活性降低12.46%；W83侵入后72h,细胞增殖活性降低10.47%,细胞生长增殖活性明显受抑制(P<0.05)。P. gingivalis ATCC 33277和W83的增殖抑制作用无显著性差异(P>0.05)；P. gingivalis侵入改变细胞周期分布,使G1期细胞增加(P<0.05),细胞周期在G1期发生阻滞,P. gingivalis W83使细胞周期发生改变的时间(24h)早于ATCC 33277 (48h); P. gingivalis侵入后24h即诱导凋亡细胞的产生(P<0.05)。P. gingivalis W83侵入后72h,死亡细胞明显增加(16.407%),与ATCC 33277侵入组(11.973%)比较有显著差异(P<0.05)。荧光显微镜观察证实凋亡细胞的存在；阶梯状电泳图谱分析显示P.gingivalis W83作用后24h和48h,HUVEC的核DNA呈现明显的拖尾条带,形成典型的DNA ladder.2、牙龈卟啉单胞菌侵入对血管内皮细胞粘附分子表达的的影响P. gingivalis ATCC 33277和W83侵入后4h即诱导ICAM-1、VCAM-1和E选择素mRNA表达(P<0.05)。ICAM-1和VCAM-1 mRNA表达水平在P. gingivalis侵入后8h达高峰(P<0.05),这种高表达可持续到P. gingivalis侵入后24h(P<0.05)。E选择素mRNA表达在P. gingivalis侵入后4h达高峰(P<0.05),8h显著回落(P<0.05),至24h时接近基础表达水平(P>0.05)；P. gingivalis ATCC 33277和W83侵入后4h即诱导ICAM-1、VCAM-1和E选择素膜蛋白表达(P<0.05),ICAM-1和VCAM-1蛋白表达在侵入后8h和24h有持续的高表达(P<0.05)；E选择素蛋白表达在侵入后8h达高峰,侵入后24h仍有较高的表达(P<0.05)；P. gingivalis W83诱导ICAM-1、VCAM-1和E选择素nRNA和蛋白表达的能力强于ATCC 33277(P<0.05).细胞免疫荧光染色证实P. gingivalis W83侵入血管内皮细胞后8h诱导ICAM-1、VCAM-1和E选择素膜蛋白的表达。3、牙龈卟啉单胞菌感染血管内皮细胞诱导IκB降解、p38 MAPK磷酸化和NF-κB p65核移位P. gingivalis ATCC 33277感染HUVEC后60min导致IκB-α降解,感染后90minIκB-α恢复至基础水平；P. gingivalis W83感染后30min后即导致IκB-α降解,60min后IκB-α蛋白水平有轻度回升,90min后IκB-α恢复至基础水平；P. gingivalis W83感染使细胞IKB-a降解时间早于ATCC 33277. P. gingivalis ATCC 33277作用于细胞后30min后导致p38 MAPK磷酸化,60min后p38 MAPK磷酸化水平明显降低,90min后恢复至基础水平；P. gingivalis W83感染HUVEC后60min导致p38 MAPK磷酸化,90min后p38 MAPK磷酸化水平明显降低。P. gingivalis ATCC 33277感染HUVEC后60min开始激活NF-κB p65核移位,感染后90min,核移位现象更明显；W83感染HUVEC后30min即开始有NF-κB p65核移位,核移位一直持续到W83感染后90min。4、NF-κB和]p38 MAPK信号通路抑制剂在P. gingivalis感染HUVEC诱导IκB-α降解、p38 MAPK磷酸化、NF-κB p65核移位和粘附分子表达过程中的作用NF-κB抑制剂MG132(20μM)和P38 MAPK抑制剂SB203580(10μM)预处理细胞30min后,再分别与P. gingivalis ATCC 33277和W83(MOI=100：1)共同培养60min、90min和8h。Western印迹法检测IκB-α和磷酸化p38 MAPK蛋白表达变化,细胞免疫荧光染色检测NF-κB P65核移位。结果显示MG132可抑制P. gingivalis W83感染HUVEC诱导的IκB-α降解、NF-κB p65核移位和ICAM-1、VCAM-1、E-selectin mRNA和蛋白表达,对p38 MAPK磷酸化无影响；SB203580可抑制P. gingivalis W83感染导致的p38 MAPK磷酸化,对IκB-α降解、NF-κB p65核移位和ICAM-1、VCAM-1、E-selectin mRNA和蛋白表达无影响。结论1、P. gingivalis侵入血管内皮细胞可抑制其增殖,使细胞周期在G1期细胞发生阻滞,并诱导细胞凋亡及ICAM-1、VCAM-1、E选择素mRNA和蛋白高表达的AS样改变,在AS炎症病理反应中有重要的意义。有效的牙周治疗、积极的控制口腔感染为我们提供了一条减少心血管疾病发生危险的可能途径。2、P. gingivalis ATCC 33277和W83侵入血管内皮细胞诱导IκB降解、p38 MAPK磷酸化和NF-κB核移位。NF-κB系统的活化参与了P. gingivalis侵入HUVEC诱导粘附分子表达的过程,NF-κB系统在P. gingivalis感染后的全身炎症反应中发挥重要的调控作用,对其进行有效的阻断和调节,将可能控制P. gingivalis感染导致血管内皮细胞粘附分子高表达的AS样改变。3、与低毒力株P. gingivalis ATCC 33277比较,高毒力株P. gingivalis W83显示更强的细胞毒性,侵入后72h导致更多的细胞死亡P. gingivalis W83诱导血管内皮细胞ICAM-1、VCAM-1和E-selectin表达的能力强于P. gingivalis ATCC 33277,这可能与其侵袭性或致脓肿作用有关。更多还原

【Abstract】 ObjectivePorphyromonas gingivalis is an obligate anaerobic gram-negative bacterium that causes periodontal disease. Destruction of host tissues results from the action of various toxic products released by P. gingivalis, as well as from the host responses elicited against this organism. The induction of this inflammatory response can lead to gingival ulceration and local vascular changes, which have the potential to increase the incidence and severity of transient bacteremias. Based on the chronic nature of this disease, and the exuberant local and systemic host response to P. gingivalis infection, recent studies have focused on the association of P. gingivalis-mediated periodontal infection and systemic diseases. Several reports support a definite relationship between periodontal infections and certain systemic conditions including atherosclerosis and cardiovascular disease.Recent studies have identified P. gingivalis in human atheromas. In addition, preliminary data have been demonstrated that P. gingivalis infection of apoE mice produced larger atherosclerotic lesions than uninfected animals. The strength of the epidemiological and initial pathological associations of P. gingivalis with atherosclerosis can be increased by the demonstration that P. gingivalis can initiate and sustain growth in human vascular cells. It has been proposed that P. gingivalis invasion of endothelial cells may induce alterations in the endothelial cell that could exhibit atherogenic properties. A hallmark of atherosclerosis is the accumulation of bloodborne leukocytes in the inflamed tissues. This process is initiated with the binding of leukocytes to the activated endothelium via the induced expression of cell adhesion molecules, including intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and E-selectins. Since the vascular endothelium is essential for the recruitment of leukocytes during atherogenesis, studies aimed at the inflammatory activation of endothelial cells pretreated by P. gingivalis may elucidate the role of this organism in atherosclerosis.Mitogen-activated protein kinase (MAPK)-related signal transduction pathways are among the most widespread mechanisms of eukaryotic cell regulation. Specifically, p38 MAPK is strongly activated during inflammatory reactions and appears to be of specific importance during LPS-mediated signal transduction. The expression of inflammatory mediators such as cytokines or adhesion molecules relies on the activation of cytosolic transcription factors. Among the primary transcription factors, nuclear factor-kappaB (NF-κB) plays a central role in the regulation of these proinflammatory molecules. Little is known about signal transduction pathways activated in target cells upon infection with P. gingivalis.We hypothesized that invasion of P.gingivalis in human endothelial cells triggers a series of molecular and cellular events that contribute to atherogenesis. The present study was to explore the capacity of P. gingivalis to modulate cell adhesion molecules expression in human endothelial cells. We also attempted to throw light on P. gingivalis dependent activation of host cell signal transduction pathways in human endothelial cells.Materials and methods1 Bacterial strains and cell culture conditions and invasion of cells by P. gingivalisP. gingivalis ATCC 33277 and W83 were cultured in BHI broth. Human umbilical vein endothelial cell (HUVEC) line (ECV-304) was obtained from China Center for Type Culture Collection (CCTCC). Bacterial suspensions (108 cells/ml) were added to confluent HUVEC monolayers at a multiplicity of infection (MOI) of 100. For inhibitor studies, Cells were pretreated with 20μM MG132(antagonist of NF-κB) or 10μM SB203580 (a specific inhibitor of p38 MAPK) for 30minutes prior to treatment with P. gingivalis.2 Assessment of endothelial cell viability and proliferation assay Cell viability and proliferation was analyzed by MTT assay. Cell cycle and apoptosis assays were performed by flow cytometry.3 RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) analysis of cell adhesion moleculesTotal cellular RNA was extracted with the use of Trizol reagent according to the manufacturer’s instructions. The transcription of total RNA into cDNA was carried out with the use of a Transcriptor First Strand cDNA Synthesis Kit (TaKaRa, Japan). PCR was performed using ICAM-1-, VCAM-1-, or E-selectin-specific primers to identify their respective specific cDNA.4 Protein extraction and western blot analysis of role of bacterial invasion in the induction of surface-associated cell adhesion moleculesCells were lysed and the protein concentration was determined with Pierce bicinchoninic acid (BCA) protein assay kit.20μg protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 10% polyacrylamide) followed by western blot analysis as previously described.5 Immunofluorescence microscopy studies of cell adhesion moleculesImmunofluorescence studies were conducted by indirect immunolabeling on cells cultured on cover slips in plastic wells. Mouse anti-human ICAM-1, rabbit anti-human VCAM-1 and rabbit anti-human E-selectin antibodies as primary antibody were added to the blocking buffer. IgG-fluorescein isothiocyanate (FITC) antibody as the secondary antibody was added. The slides were counterstained and immunoassaying was performed.6 Western blot analysis of IκBαand phospho-p38 MAPKWestern blot analysis of cytoplasmic IκBαwas carried out with anti-IκBαrabbit IgG. p38 MAPK phosphorylation was detected by western blot analysis using the phospho-specific p38 MAPK antibodies.7 Immunofluorescence microscopy detection of NF-κB p65 nuclear translocationImmunofluorescence studies were conducted on cells cultured on cover slips in plastic wells, permeabilized with 0.1% Triton-X-100 and incubated overnight with a mouse monoclonal antibody to the p65 subunit of NF-κB, and incubated with FITC-conjugated goat anti-mouse IgG secondary antibody. Then cover slips were sealed and the cells were analyzed.8 Data and statistical analysisAll experiments were performed in duplicate wells for each condition and repeated at least three times. Data are presented as mean±SD. The data was statistically analyzed by SAS 8.12 software package for repeated measures ANOVA.Results1 Effects of P. gingivalis invasion on proliferation, cell cycle and apoptosis of HUVECWe observed that P. gingivalis invasion had a highly significant inhibitory effect on cell proliferation as compared to the control group (P<0.05). However, the inhibitory effect on cell proliferation appeared unrelated to the strains since the effect is similar between P. gingivalis ATCC 33277 and W83 (P>0.05). Invasion of P. gingivalis ATCC 33277 resulted in a 12.46% decrease in cell growth at 72 h compared with control as determined by MTT colorimetric assay. Similar to P. gingivalis ATCC 33277, invasion of P. gingivalis W83 decreased MTT staining by 10.47%. And invasion of P. gingivalis could induce apoptosis at 24 h (P<0.05). In addition, the cell cycle was arrested at G1 phase by the invasion of P. gingivalis W83 at 24 h and ATCC 33277 at 48 h P<0.05).2 Effects of P. gingivalis invasion on the levels of ICAM-1, VCAM-1 and E-selectin mRNA expression in HUVECBoth P. gingivalis ATCC 33277 and W83 induced a time-dependent increase in the mRNA of HUVEC adhesion molecules ICAM-1, VCAM-1 and E-selectin. They all began to increase at 4 h post-invasion. ICAM-1 and VCAM-1 mRNA expression in both strains stimulated HUVEC peaked at 8 h, and remained elevated up to 24 h. E-selectin mRNA expression in P. gingivalis stimulated HUVEC cells peaked at 4 h post-invasion, while declined to almost baseline at 24 h. Furthermore, the effect of P. gingivalis strain W83 was more strongly than that of strain ATCC 33277 on inducing adhesion molecules mRNA expression.3 P. gingivalis stimulates cell surface ICAM-1, VCAM-1 and E-selectin expression in HUVECThe production of ICAM-1, VCAM-1 and E-selectin response to P. gingivalis invasion was detected as early as 4 h and gradually increased up to 24 h by western blot assay. It seems that P. gingivalis W83 had a little more effect on ICAM-1, VCAM-1 and E-selectin expression than P. gingivalis ATCC 33277. Expression of ICAM-1, VCAM-1 and E-selectin on the surface of P. gingivalis-invaded HUVEC was further confirmed by fluorescence microscopy. Following 8 h incubation with P. gingivalis W83, HUVEC cultures were found to exhibit extensive cell-associated staining by using either anti-ICAM-1, VCAM-1 or E-selectin antibody compared to that in uninvaded HUVEC cultures.4 Effects of P. gingivalis on IκBαdegradation and NF-κB translocation in HUVECWestern blot analysis with both strains demonstrated a time-dependent degradation of IκBα, at maximal effects at 30min (W83) to 60min (ATCC 33277) postinfection and recovered at 90min. NF-κB activation in P. gingivalis-infected HUVEC was analyzed by immunofluorescence study. It was demonstrated both strains induced a nuclear translocation of NF-κB. Note a time-dependent increasing of fluorescence intensity in the nuclei of P. gingivalis-infected HUVEC, demonstrating the nuclear translocation of NF-κB starting at 30min (W83) to 60min (ATCC 33277) postinfection. The signal remained elevated up to 90min with both strains.5 P. gingivalis-induced phosphorylation of p38 MAPK in HUVECWe demonstrated a time-dependent enhanced phosphorylation of p38 MAPK in the presence of P. gingivalis strains. Effect peaked at 30min after stimulation with strain ATCC 33277 and decreased to almost baseline at 90min. P. gingivalis W83 induced a more prolonged effect and phosphorylation remained elevated up to 90min.6 Effects of MG132 and SB203580 on induction of cell adhesion molecules by P. gingivalisTo investigate which pathways are involved in induced cell adhesion molecules production, we examined the effects of selective inhibitors on the production of adhesion molecules. MG132 is believed to block the NF-κB/IκB pathways through selectively inhibits a proteasome that specifically degrades ubiquitinated IκB after its phosphorylation. Pretreatment of cells with MG132 for 30minutes blocked the P. gingivalis-induced degradation of IκBα. Meanwhile, SB203580, a specific p38 MAPK inhibitor was added to the HUVEC for 30minutes at concentrations known to induce selective inhibition of p38 MAPK activity prior to treatment with P. gingivalis. It appeared to suppresse p38 MAPK phosphorylation without affecting other MAPK phosphorylation. Thus, both of the inhibitors proved to be good tools in investigating the contribution of each signaling pathway to cell adhesion molecules production in HUVEC.NF-κB inhibitor was able to significantly inhibit invasion of P. gingivalis mediated induction of ICAM-1, VCAM-1 and E-selectin, whereas the p38 MAPK inhibitor had no effect. In addition, we also confirmed that the induction is dependent on NF-κB. Therefore, these results suggest that invasion of P. gingivalis can induce cell adhesion molecules expression in HUVEC through the stimulation of NF-κB signaling pathway.To confirm the roles of NF-κB/IκB and p38 MAPK on P. gingivalis-induced ICAM-1, VCAM-1 and E-selectin production in HUVEC, we examined the effects of selective inhibitors on the production of ICAM-1, VCAM-1 and E-selectin mRNA by RT-PCR with invasion of P. gingivalis. The results show that production of ICAM-1, VCAM-1 and E-selectin mRNA induced by P. gingivalis invasion was inhibited by pretreatment with MG132. These observations indicate that the ICAM-1, VCAM-1and E-selectin gene promoters are activated by NF-κB during a 24 h observation. In another word, production of cell adhesion molecules in HUVECs is stimulated by invasion of P. gingivalis, and its transcription and translation are dependent on NF-κB activation through proteasome-mediated IκB degradation.Conclusions1 P. gingivalis invasion had a significant inhibitory effect on HUVEC proliferation by G1 blockade. And invasion of P. gingivalis could induce apoptosis in HUVEC.2 Both P. gingivalis ATCC 33277 and W83 induced a time-dependent increase in the mRNA and surface protein of HUVEC adhesion molecules ICAM-1, VCAM-1and E-selectin. Invasion of P. gingivalis may induce alterations in the endothelial cell that could exhibit atherogenic properties. 3 Western blot analysis with both strains demonstrated a degradation of IκBαand NF-κB activation in HUVEC. We demonstrated a time-dependent enhanced phosphorylation of p38 MAPK in the presence of P. gingivalis.4 Invasion of P. gingivalis can induce cell adhesion molecules expression in HUVEC through the stimulation of NF-κB signaling pathway. P. gingivalis plays an important role in the pathogenesis and progression of atherosclerosis partly through upregulation of cell adhesion molecules by activation of NF-κB signaling pathway.更多还原