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Study on the mechanism of periodontal pocket formation using Matrix Metalloproteinase inhibitors

基质金属蛋白酶抑制剂牙周袋形成机制的研究

基本信息

批准号：
13672156
负责人：
YOKOYAMA Masaaki
金额：
$ 2.24万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672156/
关键词：
human gingival epithelial cell human gingival fibroblast cytotoxicity assay MMP-2 MMP-9 MMP inhibitor periodontal pocket formation keratinocyte migration ヒト歯肉繊維芽細胞歯周病原細菌歯周ポケット歯肉上皮細胞歯肉繊維芽細胞 MMP TNFα TGFβ

项目摘要

The aim of this study was to clarify the behavior of Matrix Metalloproteinases (MMPs) produced by gingival cells in relation to the mechanism of periodontal pocket formation.Cultured human gingival fibroblast (HGF), cultured human gingival epithelial cell (HE), and their culture supernatants were used for all experiments. By gelatin zymography, MMP-9 from HE and MMP-2 from HGF were detected clearly, while MMP-2 from HE were faint and MMP-9 from HGF ware detected only when HGF were stimulated by TNFα. Three kinds of hydroxamic acid based MMP inhibitors (ONO-4817, ONO-MI1-514, ONO-MI1-570) were tested on these MMP activity. First, cytotoxic assay was performed to verify the safety toward host cells. The cell viability and the cell growth of HGF and HE were not suppressed by all inhibitors at final concentration of 50μM. By gelatin zymography, it was found that all inhibitors clearly inhibited MMP-2 and MMP-9 produced by HGF or HE, and the intensity of their inhibitory effect was found in order of ONO-4817, ONO-MI1-514, ONO-MI1-570. The same effect was clarified with an experiment using a colorimetric assay kit. Further investigation revealed that all inhibitors acted in a dose dependent manner. To clarify the role of MMP derived from gingival cells during a periodontal pocket formation, we investigated the effects of MMP inhibitors on keratinocyte migration in vitro.These data suggest that MMP, especially MMP-9 may participate in a pocket formation, which is concomitant with epithelial cell migration, and also support the possibility that these MMP inhibitors could be applied to the patients with periodontal disease as preventive and/or therapeutic use.

本研究的目的是阐明牙龈细胞产生的基质金属蛋白酶（MMPs）的行为与牙周袋形成机制的关系。实验采用培养的人牙龈成纤维细胞（HGF）、培养的人牙龈上皮细胞（HE）及其培养上清液。明胶酶谱法检测到HE的MMP-9和HGF的MMP-2清晰，而HE的MMP-2微弱，HGF的MMP-9仅在TNFα刺激HGF时检测到。研究了3种羟肟酸类MMP抑制剂（ONO-4817、ONO-MI1-514、ONO-MI1-570）对MMP活性的影响。首先，进行细胞毒性试验以验证其对宿主细胞的安全性。在终浓度为50μM时，所有抑制剂均未抑制HGF和HE的细胞活力和细胞生长。明胶酶谱分析发现，所有抑制剂均能明显抑制HGF或HE产生的MMP-2和MMP-9，抑制作用强度依次为ONO-4817、ONO-MI1-514、ONO-MI1-570。使用比色测定试剂盒进行实验，澄清了同样的效果。进一步的研究表明，所有的抑制剂都以剂量依赖的方式起作用。为了阐明来自牙龈细胞的MMP在牙周袋形成过程中的作用，我们在体外研究了MMP抑制剂对角质形成细胞迁移的影响。这些数据表明，MMP，特别是MMP-9可能参与伴随上皮细胞迁移的口袋形成，也支持这些MMP抑制剂可用于牙周病患者作为预防和/或治疗用途的可能性。