ESTABLISHMENT OF A CLINICAL TREATMENT SYSTEM FOR PERIODONTAL DISEASE

牙周病临床治疗体系的建立

• 批准号: 13357016
• 负责人: YAMAMOTO Kenji
• 金额: $ 32.28万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13357016/
• 关键词: Periodontal disease; Porphyromonas gingivalis; Virulence factor; Protease; Gingipains; Specific inhibitor; Drugs for periodontal disease

Periodontal disease is a common inflammatory oral disease characterized by acute progressive lesions of periodontal tissues, excessive leukocyte infiltration, and occurrence of a characteristic microflora. Porphyromonas gingivalis is a Gam-negative anaerobic bacterium that is implicated as a major etiologic agent of some types of periodontitis. This bacterium produces a unique type of cysteine proteinases referred to as gingipains in both cell associated and secretory forms. Gingipains consist of arginine-specific cysteine proteinases (Arg-gingipains, Rgp) and lysine-specific cysteine proteinase (Lys-gingipain, Kgp). The cell-associated gingipains comprise the majority (80%) of Rgp and Kgp activities and thus believed to be responsible for the virulence of the bacterium. Accordingly, the characterization and subsequent control of the cell-associated gingipain complex are thought to be the most important promising therapeutic approaches for periodontitis and related systemic disorders i … More ncluding atherosclerosis. Furthermore, we have shown previously with various P.gingivalis mutants deficient in Rgp- and Kgp-encoding genes that both enzymes play critical roles in most of the virulence of the bacterium and thus indicated that potent inhibitors of gingipains should be useful tools to assess the contribution of their proteolytic activities to the virulence of the bacterium and to facilitate the development if new therapeutic approaches to periodontal diseases. In this research project, thus, we have purified and characterized a major form of cell-associated gingipain complex from the bacterium. The complex comprised the catalytic domains and hemagglutinin domains of both rgpA and kgp gene products. Moreover, lipopolysaccharide (LPS) and phospholipids were associated with the complex. The complex significantly degraded human type I collagen and elastin and strongly disrupted viability of human gingival fibroblasts and umbilical vein endothelial cells with an efficiency which was higher than that of the monomeric gingipains. Importantly, the native complex produced only a small amount of nitrogen dioxide, TNF-□ and IL-6 by macrophages, whereas the heat-denatured complex resulted in increased production, indicating that the functional domains of LPS are structurally masked by the complex proteins. The results indicate the importance of the complex in evasion of host defense mechanisms as well as host tissue breakdown. Furthermore, we designed and synthesized a series of peptides analogues able to inhibit either Rgp or Kgp on the basis of the cleavage site specificity of histatins by each enzyme. Among this series of compounds, we found that KYT-1 and KYT-36 had the most potent and selective inhibitory activities of Rgp and Kgp, respectively. We have also demonstrated that these inhibitors are useful in assessing to what extent the proteolytic activities of Rgp and Kgp contribute to biological activities of P.gingivalis, and indicated that they should facilitate the development of new approaches to periodontal diseases. Less

牙周病是一种常见的口腔炎症性疾病，其特征是牙周组织的急性进行性病变、过度白细胞浸润和特征性微生物区系的发生。牙龈卟啉单胞菌（Porphyromonas gingivalis）是一种革兰氏阴性厌氧菌，是某些类型牙周炎的主要病原体。这种细菌产生一种独特类型的半胱氨酸蛋白酶，称为牙龈卟啉菌蛋白酶，呈细胞结合和分泌形式。牙龈蛋白酶由精氨酸特异性半胱氨酸蛋白酶（Arg-牙龈蛋白酶，Rgp）和赖氨酸特异性半胱氨酸蛋白酶（Lys-gingipain，Kgp）组成。细胞相关的牙龈卟啉菌蛋白酶包含Rgp和Kgp活性的大部分（80%），因此被认为是细菌毒力的原因。因此，细胞相关牙龈菌蛋白酶复合物的表征和随后的控制被认为是牙周炎和相关系统性疾病的最重要的有前途的治疗方法， ...更多信息 包括动脉粥样硬化。此外，我们以前已经用Rgp和Kgp编码基因缺陷的各种牙龈卟啉单胞菌突变体表明，这两种酶在细菌的大多数毒力中起关键作用，因此表明牙龈卟啉单胞菌蛋白酶的有效抑制剂应该是有用的工具，以评估其蛋白水解活性对细菌毒力的贡献，并促进牙周疾病新治疗方法的开发。因此，在本研究项目中，我们已经从细菌中纯化并表征了细胞相关牙龈菌蛋白酶复合物的主要形式。该复合物包括rgpA和kgp基因产物的催化结构域和血凝素结构域。此外，脂多糖（LPS）和磷脂与复合物。该复合物显着降解人I型胶原和弹性蛋白，并强烈破坏人牙龈成纤维细胞和脐静脉内皮细胞的活力，其效率高于单体牙龈蛋白酶。重要的是，天然复合物仅通过巨噬细胞产生少量的二氧化氮、TNF-α和IL-6，而热变性复合物导致产量增加，表明LPS的功能结构域在结构上被复合物蛋白质掩蔽。结果表明，在逃避宿主的防御机制，以及宿主组织分解的复杂的重要性。此外，我们设计并合成了一系列的肽类似物能够抑制Rgp或Kgp的基础上的裂解位点特异性的组胺每一种酶。在这一系列化合物中，我们发现KYT-1和KYT-36分别对Rgp和Kgp具有最强和选择性的抑制活性。我们还证明了这些抑制剂在评估Rgp和Kgp的蛋白水解活性对牙龈卟啉单胞菌生物活性的贡献程度方面是有用的，并表明它们应该促进牙周疾病新方法的开发。少

项目成果

組織細胞工学 第27巻

组织和细胞工程第 27 卷

• 发表时间: 2001
• 作者: 山本健二;馬場貴代
• 通讯作者: 馬場貴代

Okaji, M., et al.: "The regulation of bone resorption in tooth formation and eruption processes in mouse alvelar crest devoid of cathepsin K"J.Pharmacol.Sci.. 91. 285-294 (2003)

Okaji, M., et al.：“缺乏组织蛋白酶 K 的小鼠牙槽嵴中牙齿形成和萌出过程中骨吸收的调节”J.Pharmacol.Sci.. 91. 285-294 (2003)

Ueshima J., e al.: "Dps from the obligate anaerobe Porphyromonas gingivalis : purification, gene cloning, gene expression and mutant studies"Infection and Immunity. (in press). (2003)

Ueshima J.等人：“来自专性厌氧菌牙龈卟啉单胞菌的Dps：纯化、基因克隆、基因表达和突变体研究”感染和免疫。

Nakanishi H., et al.: "Involvement of nitric oxide released from microglia-macrophages in pathological changes of cathepsin D-deficient mice"Journal of Neuroscience. 21. 7526-7533 (2001)

Nakanishi H.等人：“小胶质细胞-巨噬细胞释放的一氧化氮参与组织蛋白酶 D 缺陷小鼠的病理变化”神经科学杂志。

歯周病とジンジパイン

牙周病和牙龈疼痛

• 发表时间: 2003
• 期刊: 日本薬理学会誌 122
• 作者: Kadowaki;T. et al.;Hasegawa Y. et al.;Moriguchi S. et al.;Tsukuba T. et al.;Kadowaki T. et al.;山本健二;筑波隆幸;門脇知子
• 通讯作者: 門脇知子