Effects of Melatonin on regenerative treatment in periodontal disease

褪黑素对牙周病再生治疗的影响

• 批准号: 13672194
• 负责人: FUJII Takeo
• 金额: $ 1.92万
• 依托单位: Health Sciences University of Hokkaido
• 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 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672194/
• 关键词: melatonin; fibroblast; IL-8; phosphophoryn; rhBMP-2; bone formation; 実験的歯周炎; LPS; CD14; Anti-CD14 antibody; IL-1β

(1) Prostaglandin E2 (PGE2), a potent mediator of inflammation, has been detected in gingival crevicular fluid. The aim of this study was to examine the modulating effect of PGE2 on LPS-induced IL-8 production by human gingival fibroblasts (HGFs) in vitro. HGFs were obtained from medically healthy donors who were clinically free of periodontal disease. Cells were suspended in OMEM supplemented with 5% heat-inactivated FCS, seeded at a cell density of 1x104 cells/well in 96-well microtiter plates and incubated at 37oC in 5% C02 for 3 days. Culture medium was removed and replaced with DMEM containing 1% FCS 24 h prior to the experiments. HGFs were then exposed to various concentrations of LPS from Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans or Escherichia coli. The effect of PGE2 was studied by exposing HGFs cultures to various concentrations of PGE2 for 2 h before adding LPS preparations. Following incubation, culture supernatants were harvested and assayed for IL-8 u … More sing an ELISA kit. The expression of IL-8 mRNA, after stimulation with PGE2, was determined by RT-PCR. IL-8 production by HGFs was shown to be related to the time of incubation and the dose of LPS. A slight increase in IL-8 levels was observed when cells were exposed to PGE2 alone compared to controls. When HGFs were pre-stimulated with PGE2 and then exposed to LPS, IL-8 production was increased two to three times compared to non-treated cells. Herbimycin A completely abolished the secretion of IL-8. These results indicate that PGE2 can modulate cytokine secretion from HGFs and increase IL-8 production from cells exposed to LPS. IL-8 production induced by LPS was dependent on the tyrosine phosphorilation pathway.(2) Objective: To determine whether phosphophoryn, major non-collagenous protein in dentin, plays a role of a co-factor of rhBMP-2 when rhBMP-2 induces hard tissue formation in vivo. Methods: Atelocollagen absorbed to porous hydroxyapatite was used as the carrier of rhBMP-2 for induction of ectopic bone formation. Five microgram of rhBMP-2 and 1 mg of phosphophoryn were added to apatite/ collagen composite. Then rhBMP-2/apatite/collagen/phosphophoryn was implanted subcutaneously into male Wistar rats (4 week old) at bilateral sites in the back. RhBMP-21 apatite/collagen or rhBMP-2/apatite was also implanted as controls. Rats were sacrificed and the implants were extirpated, at weekly intervals for 3 weeksafter implantation. The implants were analyzed histologically and biochemically. Results: Bone formation was observed at some small parts in all implants 1 week after implantation (week 1 implants). Bone formation by rhBMP-2 combined with apatite/ collagen/ phosphophoryn was more than that by rhBMP-2 combined with apatite/ collagen at week 2. Furthermore, bone was formed at the surface area of the implant, and bone remodeling occurred at week 3. Bone marrow cells took the place of grains of hydroxyapatite at the central part in rhBMP-2/ apatite/ collagen/ phosphophoryn. ALPase activity of rhBMP-21 apatite/ collagen/ phosphophoryn was about two times more than that of rhBMP-2/ apatite/ collagen at week 3. Conclusions: It was indicated that phosphophoryn acts as an accelerate factor of rhBMP-2 for induction of ectopic bone formation, and was suggested that rhBMP-2/ apatite/ collagen/ phosphophoryn composite could be an excellent biomaterial in bone and tooth reconstruction. Less

(1) 前列腺素 E2 (PGE2) 是一种有效的炎症介质，已在龈沟液中检测到。本研究的目的是检查 PGE2 对 LPS 诱导的人牙龈成纤维细胞 (HGF) 产生 IL-8 的调节作用。 ) 体外，HGF 取自临床上无牙周病的健康捐献者，细胞悬浮在补充有 5% 的 OMEM 中。热灭活的FCS，以1x104个细胞/孔的细胞密度接种在96孔微量滴定板中并在37℃下在5％CO 2 中培养3天，在接种前24小时除去培养基并更换为含有1％FCS的DMEM。然后将 HGF 暴露于来自牙龈卟啉单胞菌、伴放线放线杆菌或的不同浓度的 LPS。通过将 HGF 培养物暴露于不同浓度的 PGE2 中 2 小时，然后添加 LPS 制剂来研究大肠杆菌的作用，然后收获培养物上清液并使用 ELISA 试剂盒测定 IL-8 的表达。用 PGE2 刺激后，通过 RT-PCR 测定 IL-8 mRNA，结果显示 HGF 产生的 IL-8 与孵育时间和 LPS 剂量相关。与对照组相比，当细胞单独暴露于 PGE2 时，观察到 IL-8 水平略有增加。当用 PGE2 预刺激 HGF，然后暴露于 LPS 时，与未处理的相比，IL-8 的产量增加了两到三倍。 Herbimycin A 完全消除了 IL-8 的分泌，这些结果表明 PGE2 可以调节 HGF 的细胞因子分泌，并增加暴露于 LPS 诱导的细胞的 IL-8 产生。 (2)目的:探讨牙本质中主要的非胶原蛋白磷酸蛋白在rhBMP-2诱导体内硬组织形成过程中是否发挥辅助因子的作用。多孔羟基磷灰石用作rhBMP-2的载体，用于诱导异位骨形成。5微克的rhBMP-2和1mg的rhBMP-2。将rhBMP-2/磷灰石/胶原/磷灰石添加到雄性Wistar大鼠（4周龄）背部的双侧部位的rhBMP-21磷灰石/胶原或rhBMP-2/磷灰石中。还植入了大鼠作为对照，并在植入后每周一次将植入物摘除。对植入物进行分析。组织学和生化结果：植入后1周（第1周种植体）在所有种植体的一些小部位观察到骨形成，rhBMP-2联合磷灰石/胶原蛋白/磷蛋白的骨形成多于rhBMP-2联合的。第2周时磷灰石/胶原蛋白。此外，在植入物的表面区域形成骨，并在第3周发生骨重塑。骨髓细胞取代了骨颗粒rhBMP-2/ 磷灰石/ 胶原蛋白/ 磷酸蛋白中心部分的羟基磷灰石在第3 周时，rhBMP-21 磷灰石/胶原蛋白/ 磷酸蛋白的ALPase 活性大约是rhBMP-2/ 磷灰石/ 胶原蛋白的两倍。表明磷酸化蛋白是 rhBMP-2 诱导异位骨形成的加速因子，并提出 rhBMP-2/磷灰石/胶原蛋白/磷酸复合物可能是骨骼和牙齿重建的优良生物材料。

项目成果

P.L.Wang, M.O.Mori, T.Fuji, Y.Kowashi et al.: "Effect of Anti-CD14 Antibody on Experimental Periodontitis Induced by Porpyromonas gingivalis Lipopolysaccharide"Jpn. J. Pharmacol.. 89. 176-183 (2002)

P.L.Wang、M.O.Mori、T.Fuji、Y.Kowashi等：“抗CD14抗体对牙龈卟啉单胞菌脂多糖诱导的实验性牙周炎的作用”Jpn。

T.Saito, F.Kobayashi, T.Fujii, K.Bessho: "Effect of phosphophoryn on rhBMP-2-induced bone formation"Arch Oral Biology. 49. 239-243 (2004)

T.Saito、F.Kobayashi、T.Fujii、K.Bessho：“磷酸化蛋白对 rhBMP-2 诱导的骨形成的影响”Arch Oral Biology。

Pao-Li Wang, Maro Oido-Mori, Takeo Fujil, Yusuke Kowashi, Masano Kikuchi, Yasushi Suetsugu, Junzo Tanaka, Yasutaka Azuma, Mitsuko Shinohara, Kiyoshi Ohura: "Effect of Anti-CD14 Antibody on Experimental Periodontitis Induced by Porphyromonas gingivalis lip

Pao-Li Wang、Maro Oido-Mori、Takeo Fujil、Yusuke Kowashi、Masano Kikuchi、Yasushi Suetsugu、Junzo Tanaka、Yasutaka Azuma、Mitsuko Shinohara、Kiyoshi Ohura：“抗 CD14 抗体对牙龈卟啉单胞菌引起的实验性牙周炎的影响

P.L.Wang, O.Mori, T.Fujii, Y.Kowashi et al.: "Effect of Anti-CD14 Antibody on Experimental Periodontitis Induced by Porpyromonas gingivalis Lipopolysaccharide"JPn.J.Pharmacol. 89. 176-183 (2002)

P.L.Wang、O.Mori、T.Fujii、Y.Kowashi 等：“抗 CD14 抗体对牙龈卟啉单胞菌脂多糖诱导的实验性牙周炎的效果”JPn.J.Pharmacol。

T.Saito, F.Kobayashi, T.Fujii, K.Bessho: "Effect of phosphophoryn on rhBMP-2-induced bone formation"Arch Oral Biology. 49. 239-243 (2004)

T.Saito、F.Kobayashi、T.Fujii、K.Bessho：“磷酸化蛋白对 rhBMP-2 诱导的骨形成的影响”Arch Oral Biology。