【作者】 郭威；

【导师】 杨丕山； Neil Hunter； 叶萍；

【作者基本信息】 山东大学 ， 口腔临床医学（专业学位）， 2015， 博士

【摘要】 研究背景牙周病(periodontal disease)是目前世界公认的受局部因素和全身因素相互促进、相互拮抗而产生的一类机制复杂的多因素疾病。慢性牙周炎(chronic periodontitis)是其中最常见的一类,其典型症状表现为牙龈的炎症和出血,牙周袋的形成,牙槽骨的吸收、以及最终牙齿的松动和脱落。口腔牙龈上皮作为抵御外界细菌感染的第一道屏障,维持着牙龈组织的健康和机体的平衡。健康的牙龈上皮可分为三部分：口腔上皮,龈沟上皮和结合上皮。非角化的龈沟生皮和结合上皮作为第一道屏障与龈沟内细菌紧密接触。牙龈的上皮细胞可以通过感受病理识别受体的刺激,释放前炎症因子、细胞因子和防御素,进而破坏上皮紧密连接结构而向深部移行形成深沟或深袋,最终形成牙周袋。这些变化通常是受感染上皮的不同细菌产物,特别是牙龈卟啉单胞菌的作用造成的,其内的微生物膜释放的细菌产物可以破坏宿主的免疫反应。在众多的多蛋白炎性复合物中，炎症复合小体在天然免疫中占有重要作用,这类复合体由NLR家族成员组成,包括3个亚类,NODs, NLRPs, IPAP,在这几个亚类中,NLRP3炎症复合物是目前研究最多的一类。NLRP3炎症复合物由NLRP3, ASC,Caspase-1组成,激活后释放IL-1β,IL-18前炎症因子,这是在宿主防御和感染中反应最为明显的两个因子。本研究中,我们将探讨牙龈上皮细胞系在不同因素刺激作用下NLRP3炎症复合物的反应机制及口腔牙龈上皮细胞紧密连接与炎症反应间的关系,进而为有效的控制和治疗牙周病提供理论依据。方法1.样本采集：选取符合实验条件预备拔牙的牙周病患者共26名,详细记录临床及X线片资料。按照实验要求切取牙龈组织后做冰冻切片及组织染色。2.口腔上皮细胞培养实验用H413细胞系是从口腔鳞状上皮细胞癌中分离出具有复层上皮细胞形态并高表达CD24的一类细胞系。克隆细胞系培养条件是JMEM,胎牛血清和盘尼西林/链霉素的混合培养基,37℃含5%C02的孵箱。该细胞系一般3天进行一次传代。3.牙龈卟啉单胞菌培养牙龈卟啉单胞菌ATCC33277培养于专用的细菌厌氧箱中,当细菌从-80℃复苏后接种于TSB培养皿上,待生成单菌株后,挑去单颗置于eTSB(含维生素K)的培养液中,测OD值达1.0时用于实验。4.免疫组化染色和共聚焦激光扫描显微术所有切片及细胞使用免疫组化染色,共聚焦激光显微镜(Olympus FV 10-MCPSU (405nm,473nm,633nm), NTT electronic Optiλ, (559 nm)激光)观察并拍照,由两个观察员独立观察,标记,分类,读数并记录。所有图片用共焦采集软件(FV10-ASW1.7)处理并保存为TIF文件。5.提取细胞RNA,转录合成cDNA,实时定量RT-PCR:用Trizol提取法(Trizol regent, Invitrogen)提取细胞总RNA。用SuperscriptⅢ反转录试剂盒(Invitrogen)实时定量RT-PCR测在牙龈卟啉单胞菌感染、LPS以及ATP刺激的不同时间时细胞炎症复合物的基因表达水平以及细胞紧密连接中各种基因的表达水平的差异。6.酶联免疫吸附法检测IL-1p及IL-18的表达水平：在上述处理条件中,不同时间点(0,1,2,3,4,5,6个小时)收集细胞培养盘中的media离心取上清按照ELISA kits (Abnova, Taiwan)设计96孔盘,分析IL-1p及IL-18的蛋白表达水平。7.免疫印迹法分析蛋白表达水平：按实验设计处理细胞后,收集蛋白,加热,变性,固定,加样,跑胶,转膜,封闭,常温,过夜。一抗孵育4小时,二抗孵育2小时,终止反应后观察。8.统计分析：Prism 3.03软件进行统计分析。P<0.05具有统计学差异。结果：1.牙周活组织检查中NLRP3和occludin的表达分布健侧和患侧的龈沟上皮以及健侧和患侧的袋内上皮之间NLRP3的表达有显著性差异。健侧牙龈的龈沟上皮和结合上皮与患侧袋内上皮之间occludin有显著性差异。2.Pg、LPS、ATP+Pg、ATP+LPS作用后炎症复合物的表达牙龈卟啉单胞菌刺激细胞抑制NLRP3炎性复合物的合成和释放。LPS促进NLRP3,ASC而非caspase-1的表达。ATP可以促进NLRP3炎性复合物的释放和IL-1β及IL-18细胞因子的生成。IL-1β在牙龈卟啉单胞菌作用下出现明显表达增高,而LPS对它的作用并不明显。相反,牙龈卟啉单胞菌感染并不能诱导IL-18的合成,而LPS或ATP的刺激可以诱导IL-18的合成和释放。3. Pg、LPS、ATP+Pg、ATP+LPS作用后细胞紧密连接的表达牙龈卟啉单胞菌作用后,仅出现Claudin-4的短暂升高,而Occludin、JAM-A、 Claudin-1、Claudin-15及ZO-1均表现出了明显的抑制和下调。LPS作用后,除Occludin、JAM-A、claudin-4之外,其余的蛋白均出现了明显的上调。ATP作用后Occludin、JAM-A、Claudin-1、Claudin-4、Claudin-15及ZO-1的表达一过性增强。结论：1.牙龈卟啉单胞菌完全抑制了NLRP3炎性复合物的表达过程,而且除Claudin-4外,其他发挥屏障功能的蛋白均出现失调状态。这为细菌等致病因素进一步感染细胞提供了便利。2.LPS促进NLRP3,ASC而非caspase-1的表达增强。Occludin、JAM-A和claudin-4作为第一道屏障,在受到LPS的刺激时表现出抑制作用,提示LPS在促进炎症反应过程及破坏前沿细胞屏障防御系统中具有重要作用。3.ATP促进NLRP3炎性复合物释放、细胞紧密连接蛋白表达增强和IL-1β及IL-18细胞因子的生成。这提示ATP在刺激细胞膜极性化并导致细胞膜完整性的破坏及细胞内离子浓度的变化的过程中具有重要作用。4. Cspase-1通常以非活化的酶原状态存在。在LPS刺激后并没有任何反应直到ATP活化细胞之后才释放。说明它的激活严格受到炎症复合物的调控。5.IL-1β作为天然免疫过程中最早释放的细胞因子之一,在参与激活和免疫调节过程中具有重要的作用。更多还原

【Abstract】 Back groundPeriodontal diseases are the main chronic infectious diseases of the oral cavity, which can affect the periodontal supporting tissues and/or result in the breakdown of the tooth-supporting tissues, even ultimately lead to resorption of the alveolar bone and tooth loss. Among these diseases, chronic periodontitis is the most common and prevalent chronic infections in humans worldwide.The gingival epithelium is a very important stratified squamous epithelium that interfaces between the external environment and prevents itself to be destroyed by the stimuli from outside, which can be classified into three types:the oral gingival epithelium; the sulcular epithelium and the junctional epithelium. The sulcular and the coronal margins of the junctional epithelium are in close contact with bacteria in the gingival sulcus and appear to be crucial sites with regard to the development of periodontal diseases. When the inflammation happened, amount of pathogenic microorganism including the periodontal pathogen, Porphyromonas gingivalis(Pg) begin to produce a broad array of potential virulence factors involve in tissue colonization and destroy the junction epithelium and the epithelial cells, which can sense and respond to the presence of bacteria, then make the epithelial barrier disappear, connective tissue attachment loss, bone resorption and finally convert to a pocket epithelium.The NLR family is an intracellular multiprotein complex containing different combinations of proteins, named’inflammasome’ and includes NODs, NLRPs, IPAP inflammasomes. Among these inflammasomes, NLRP3 inflammasome is the best-characterized multi-protein complex composed of the NLRP3, the ASC, and the caspase-1, which can recognize the pathogen- or danger-associated molecular patterns (PAMPs or DAMPs) and induces the maturation and secretion of IL-1β and the IL-18, which further lead to the recruitment of other immune cells to the affected cells or tissues and result in chronic inflammation and subsequent tissue damage.In this study, we will examine the inflammasome responses of the oral epithelial cell-culture model(H413 epithelial cell line) and try to elucidate this correlation with the affected periodontal tissues and tight junction proteins, which contributes to the further investigations into the immune mechanisms of NLRP3 inflammasome between bacteria and gingival epithelium and useful information for prevention and therapy of Periodontitis.Methods1. Tissue samplesGingival tissues were obtained with Ethics Committee approval and informed consent from 26 adult patients attending the periodontal clinic. Subjects had done the examination and X-ray strictly. Slides with sequential sections were stored in sealed boxes at -70℃ until required for immunostaining and confocal laser scanning microscopy.2. Oral epithelial cell cultureThe epithelial cell line (H413) derived from a human oralsquamous cell carcinoma displays stratified epithelialcell morphology and high CD24 expression in culture. The cloned cells were cultured in Eagle’s minimum essential medium (JMEM, Joklik modification, Sigma-Aldrich), penicillin/streptomycin(100 IU/ml, Sigma), and 10% fetal calf serum (FCS, CSL Limited, Victoria, Australia) at 378℃ in 5% CO2 Cultures were harvested with triple express (replacement for trypsin, Invitrogen, Australia) in PBS and sub-cultured every 3 days.3. Porphyromonas gingivalis culturePorphyromonas gingivalis (ATCC 33277) was cultured anaerobically for 24 h at 37℃ in a trypticase soy broth supplemented with haemin (5 mg/ml, Sigma) and menadione (1 mg/ml, Sigma). On the day of cell treatment, bacteria were centrifuged at 5000 rpm, and 4℃ for 15 min, washed twice and re-suspended in cold PBS, pH 7.3.4. Immunostaining and confocal laser scanning microscopyAll slides and cells were were treated with immunostaining. Confocal images were obtained with an Olympus Fluoview (FV) 1000 equipped with Olympus FV 10-MCPSU (405 nm,473 nm,633 nm) and NTT electronic Optil (559 nm) lasers. All fluorescence images prepared with confocal acquisition software (FV10-ASW 1.7) were stored and exported as TIF files.5. RNA isolation and quantitative real-time RT-PCRCells were harvested in 1ml of Trizol reagent (Invitrogen) and RNA extracted as per the Trizol protocol. For reverse transcription, the First-Strand cDNAs were synthesized with SuperScriptTM III Reverse Transcriptase (Invitrogen) according to the manufacturer’s (Invitrogen) protocol.6. Immunoassay-ELISA to quantify levels of IL-1β and IL-18A standard sandwich enzyme-linked immuno-sorbent assay (ELISA) was used to measure cytokine production of IL-1β and IL-18. cell cultures were collected at each hour (1,2,3,4,5,6 h), then particles removed by centrifugation and analysed immediately or aliquoted and stored at -20℃.7. Immunoblots for NLRP3, ASC and caspase-1 proteins and tight junction proteinsProteins of the different conditions were extracted in SDS sample buffer and separated by PAGE using gradient 5% to 12% mini-gels, transferred to nitrocellulose membranes (Bio-Rad) and blocked overnight with 3% BSA (Sigma) in 0.1 M Tris buffered salts solution pH 7.4 (TBS). Data were analysed after development of reactivity for proteins from control antibody.8. Statistical AnalysisAll data were analysed by Prism 3.03 software. paired t-test (mean ± S.D., two-tailed,95% CI range) was used for real-time RT-PCR and ELISA. P< 0.05 was considered statistically significant.Results1. Expression of NLRP3 and occludin in periodontal biopsiesComparison of minimally inflamed and periodontitis groups indicated statistically significantly different patterns to NLRP3 and occludin were observed between SE (Min/Perio) and JE/PE (Min/Perio).2. Inflammasome expression in response to P. gingivalis, LPS and ATP plus P. gingivalis / LPSThere was down-regulation of NLRP3 gene expression after P. gingivalis infection at 2 and 4 h compared with the control group. LPS elicits a striking immune response through up-regulation of the gene expression of NLRP3 and ASC, but not caspase-1. ATP activates the NLRP3 inflammasome, subsequently releasing cytokines IL-1β and IL-18. Meanwhile, gene expression of IL-1β up-regulated after P. gingivalis infection.3. Cytokines expression in response to P. gingivalis, LPS and ATP plus P. gingivalis / LPSThere was only up-regulated Claudin-4 gene expression after P. gingivalis infection compared with the control group. LPS elicits a striking immune response through up-regulation of the gene expression of Claudin-1、Claudin-15 and ZO-1. ATP activates the gene expression of Occludin、JAM-A、Claudin-1、Claudin-4、Claudin-15 and ZO-1.Conclusion1. P. gingivalis infection down-regulates NLRP3 inflammasome components and tight junction proteins except Claudin-4 in epithelial cells, which helps bacteria to infect cells more deeply.2. LPS elicits a striking immune response through up-regulation of the gene expression of NLRP3 and ASC, but down-regulation of the gene expression of Occludin、JAM-A and claudin-4, which indicates that LPS would be a key factor in eliciting the inflammatory response that leads to the diseased state and is considered an important virulence factor in destroying the first defense barrier of gingival epithelia cells.3. ATP activates the NLRP3 inflammasome, tight junction proteins, subsequently releasing cytokines IL-1β and IL-18, which support that extracellular ATP, as a danger signal, results in form of membrane pores that induce damage of membrane integrity or cause perturbation of the intracellular ion concentration.4. Caspase-1 is synthesised as an inactive zymogen. Its activation is inhibited after LPS stimulation until activated with ATP stimuli, which means caspase-1 is tightly regulated by inflammasomes.5. IL-1β is a critical cytokine in the host defence and its secretion in the early stages of P. gingivalis infection would play a very important role in combating the invading pathogen as part of the innate immune response.更多还原