【作者】 毛松；

【导师】 何安光；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2002， 博士

【摘要】 目的 牙龈卟啉单胞菌(Porphyromonas gingivalis，Pg)是G~-、产黑色素的厌氧短杆菌，是牙周炎、尤其是慢性成人牙周炎和早发性牙周炎最主要的优势菌之一，是目前公认的牙周炎病原菌。很多研究显示慢性成人牙周炎的进程与病变部位龈下菌斑中Pg的存在密切相关，Pg影响牙周组织细胞多种炎性介质的表达，这些介质关系到牙周炎症的启动和发展。然而，Pg的致病机理还不十分清楚，目前的研究表明，Pg具有多种毒力因子，包括菌毛、外膜蛋白、蛋白酶、脂多糖(lipopolysaccharide，LPS)等，这些毒力因子可以通过多种途径逃脱宿主的防御系统，破坏宿主的牙周支持组织。探讨Pg诱发牙周炎性反应和牙周组织破坏的病理机制，确定Pg主要的效应位点，可以为牙周疾病的免疫治疗奠定理论基础，在牙周病学的基础研究中有重大的意义。 慢性成人牙周炎的病理学特征是白细胞在牙龈结缔组织中的大量浸润，白细胞在成人牙周炎的发生和发展过程中具有不可忽视的作用，而其活性的发挥依赖着一系列粘俯分子介导的与细胞和细胞外基质的接触和粘附。目前，粘俯分子因其广泛的作用而受到人们普遍的关注，特别是在炎性反应中白细胞的附壁和移行中的作用更是人们关注的热点。细胞间粘附分子-1(intercellular adhesion molecule 1，ICAM-1)和血管细胞粘附分子-1(vascular cell adhesion molecule 1，VCAM-1)是表达于细胞表面的粘附分子家族，涉及到白细胞的聚积和激活，是炎性反应中重要的前炎性介质；而白细胞介素8(interleukin 8，IL-8)和单核细胞趋化蛋白-1(monocyte chemoattractant protein 1，MCP-1)分别是中性粒细胞和单核细胞的趋化因子，可以选择性地诱导白细胞向炎症部位聚积，对炎性反应中白细胞的浸润和分布起重要的调节作用。很多研究显示Pg影响牙龈组织细胞表达ICAM-1、VCAM二＊ 和MCPl，这在牙周炎症的病理机制中有重要的意义。血管内皮细胞是对炎性介质、LPS和细菌刺激都非常敏感的细胞，牙周病原菌对血管内皮细胞的作用一直是牙周病学研究的重要内容。然而，关于内对内皮细胞表达 ICAM士、ILS和 MCP－l的报道在国内外都不多见。本实验观察了 Pg对人脐静脉血管内皮细胞（hUInan umbilical vein endothelial cells，HUVEC）表达ICAMl、ILS和MCP－l的影响，并分析杆作用可能的效应部位，比较Pg毒力株W83和非毒力株ATCC33277间的差异，同时观察慢性成人牙周炎患者牙龈组织ICAM＊和VCAM上表达的改变，以深入探讨Pg诱发牙周疾病的可能机制。 方 法 1．细菌培养：Pg W83株和 ATCC33277株被培养在心脑浸液培养基中，补充以酵母浸膏5 g／L、氯化血红素5 mgiL、维生素民lmgiL。培养过程在厌氧袋中进行，温度35℃，直至到达理想的浓度、利用Gram染色法和羊血琼脂板培养法检测Pg的纯度。经7000xg，10而n离心后，将Pg以10’CFU／Inl的浓度悬浮于无菌的磷酸盐缓冲液（PBS）中，备用。 2．细胞培养：原代的 HUVEC来源于新鲜的人脐静脉刃．二％胶原酶处理脐静脉，收集细胞，离心后置于胶原包被的100－mm的培养皿中，培养液为补充 30 mg／L内皮细胞生长补体、含 20％胎牛血清（FCS）的 M199。HU－VEC被置于 37tj％CO。培养箱中培养至形成单层对．025％胰蛋白酶一EDTA处理后系列传代，3至5代的HUVEC用于实验。内皮细胞的起源性由 AC－LDL摄人实诞实。 3．HUVEC和 Pg的相互作用：先于共同培养，HUVEC被置于胶原包被的12孔培养板中，37℃J％CO。培养箱中过夜，使细胞贴壁成单层。将10’CFU／Inl的 Pg悬液sIJ用紫外线＊0 W，10 cm，5 dn入超声波③ dn，5次）和热处理K6℃，30 dn）灭活，用20％FCS的M199稀释为系列浓度，用以刺激HUVEC＊ 矿L的大肠杆菌LPS用作阳性对照，不含作的20％FCS M199中的 HUVEC做阴性对照，共同培养一定时间后收集细胞和培养上清，利用流式细胞仪测定HUVEC表面ICAM＊阳性细胞百分数和阳性细胞表达强度，80℃保存培养上清，用于测定ILS和MCP－l的含量。 ·2· 另外，用125，250，500，100 U／d的多粘菌素B预处理1吟L粉或 10儿FtU／nd的 Pg，另将含 2000 U／nd TNF心上 mgiL LPS和 10‘cFU／dPg置于水中煮沸 15 mn，用20％FCS M199 10倍稀释，分别用预处理的Pg、LPS或 TNF心刺激 HUVEC，未处理的 Pg、LPS和 TNF心做对照，阴性对照为不含刺激物的M199培养液中的HUVEC，共同培养20h后收集细胞，以观察多粘菌素 B和热处理对 Pg诱导 HUVEC表达 ICAM＊o4和MCP＊的影响。 4．流式细胞技术分析 HUVEC表面 ICAM＊的表达：HUVEC和 Pg或LPS、TNF心共同培养一定时间后，用 0．025％胰蛋白酶E删处理并收集细胞，含lmgiL牛血白蛋白的PBS洗细胞，加荧光标记的特异性鼠抗ICAM－1单克隆抗体 10 gi，4t暗室中孵育 30 dn。清洗后悬浮于 800 giPBS中，流式细胞技术测定ICAM＊阳性细胞百分数和阳性细胞表达强度。 5．酶联免疫吸附实验尸nZym－linked immunosorbent assny，ELISA）测?更多还原

【Abstract】 ObjectivesPorphyromonas gingivalis (P. gingivalis) is thought to be one of the most important pathogens in periodontitis, especially in chronic adult periodontitis and early-onset periodontitis. Many reports showed that the progress of chronic adult periodontitis is closely related with P. gingivalis , which exists in subgingi-val plaque in periodontitis patients. P. gingivalis influences the expression of many inflammatory cytokines in various cells in periodontal tissue, and these cy-tokines are very important for initiation and development of periodontitis. However, we know a little on the pathogenesis of periodontitis induced by P. gingivalis . A body of research revealed that P. gingivalis processes many virulent factors, including fimbri, out-membrane protein, proteinase, lipopolysac-charide (LPS) and so on. These virulent factors could escape the defense system in many ways to destroy periodontal supporting tissue. It is significant to investigate the pathogenesis of P. gingivalis in inducing inflammatory and immuno-logical reaction in periodontitis and determine the effective domain of P. gingivalis since it will provide the theoretical basis for immunotherapy in periodontitis.One of the pathological characters of adult periodontitis is the accumulation of leukocytes in gingival connective tissue. Leukocytes play an important role inthe process of periodontitis, and their activity is dependent on the contact and adhesion with cells and extracellular matrix mediated by a serial of adhesion molecules. At present, adhesion molecules are becoming the hot topic for their extensive functions. Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 ( VCAM-1) are an adhesion molecule family expressing on the surfaces of many kinds of cells. They are important pro-inflammatory cy-tokines in inflammatory reaction and involved in the activation and accumulation of leukocytes. Interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) are specific chemoattrctants for neutrophils and monocytes, respectively. Many studies showed that P. gingivalis affected the expression of ICAM-1, VCAM-1, IL-8 and MCP-1 in gingival tissue and they might play an important role in the pathogenesis of periodontitis. Vascular endothelial cells are important in the research of periodontitis since they are very sensitive to cytokines, LPS and bacteria. The interaction of vascular endothelial cells and periodontal pathogens is one of the major topics in periodontal research. However, a little information of the effects of P. gingivalis on the expression of ICAM-1, VCAM-1, IL-8 and MCP-1 was reported in China, even in the world. In the present experiment , we investigated the effects of P. gingivalis on the expression of ICAM-1, IL-8 and MCP-1 in human umbilical vein endothelial cells (HUVEC) , and analyzed the possible effective domain of P. gingivalis , compared the differential effect between P. gingivalis virulent strain W83 and invirulent strain ATCC33277. In addition, we investigated the expression of ICAM-1 and VCAM-1 in gingival samples of periodontitis patients in order to find out the possible pathogenesis of periodontitis induced by P. gingivalis .Methods1. Bacterial culture: P. gingivalis strains of W83 and ATCC33277 were grown in Brain Heart Infusion Broth, supplemented with yeast-extract (5 g/L) , Hemin (5mg/L), and Vitmin K3(1mg/L). The incubation was carried out inanaerobic bag at 35 C until at an optical density. Bacterial cells were washed twice with sterile phosphate buffered saline (PBS) by centrifiigation of 7000+ g for 10 min, and suspended in PBS at a concentration of 109 CFU/ml for use.2. Endothelial cell culture: Primary HUVEC were obtained from fresh human umbilical vein. The cells were collected after the treatment of 0.1% colla-genase and were incubated in collagen-coated 100-mm flasks with M199 medium supplemented with 30 mg/L of endothelial cell growth supplement and 20% fetal calf serum (FCS). HUVEC were maintained at 371 in a 5% CO2 atmosphe更多还原