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。本研究旨在探讨PGE2对lps诱导的人牙龈成纤维细胞（HGFs）产生IL-8的调节作用。生长因子来源于医学上健康且临床无牙周病的供体。细胞悬浮在添加5%热灭活FCS的OMEM中，以细胞密度为1x104个/孔接种于96孔微滴板中，在37℃、5% co2中孵育3天。实验前24 h取出培养基，用含1% FCS的DMEM代替。然后将hgf暴露于来自牙龈卟啉单胞菌、放线菌和大肠杆菌的不同浓度的LPS中。在加入LPS制剂之前，将hgf培养物暴露于不同浓度的PGE2中2小时，研究PGE2的影响。孵育后，收集培养上清液，检测IL-8的含量。经PGE2刺激后，RT-PCR检测IL-8 mRNA的表达。hgf产生的IL-8与孵育时间和LPS剂量有关。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预先刺激hgf，然后暴露于LPS时，与未处理的细胞相比，IL-8的产生增加了两到三倍。红霉素A完全抑制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的细胞IL-8的产生。LPS诱导IL-8的产生依赖于酪氨酸磷酸化途径。(2)目的：确定牙本质中主要的非胶原蛋白phosphophoryn在体内rhBMP-2诱导硬组织形成时是否作为rhBMP-2的辅助因子发挥作用。方法：以多孔羟基磷灰石吸附的胶原为载体，诱导异位骨形成。在磷灰石/胶原复合材料中加入5微克rhBMP-2和1毫克磷酸蛋白。4周龄雄性Wistar大鼠双侧背部皮下植入rhBMP-2/磷灰石/胶原/磷酸蛋白。同时植入RhBMP-21磷灰石/胶原或rhBMP-2/磷灰石作为对照。处死大鼠，取出植入物，每隔一周取出一次，共植入3周。对植入物进行组织学和生化分析。结果：所有种植体在种植1周后（种植1周）均有少量骨形成。第2周时，rhBMP-2联合磷灰石/胶原/磷酸化蛋白成骨量大于rhBMP-2联合磷灰石/胶原成骨量。此外，在种植体表面形成骨，骨重塑发生在第3周。在rhBMP-2/磷灰石/胶原/磷酸化蛋白中，骨髓细胞取代羟基磷灰石颗粒位于中心位置。第3周时，rhBMP-21磷灰石/胶原/磷酸化蛋白的ALPase活性约为rhBMP-2/磷灰石/胶原的2倍。结论：phosphoryn可作为rhBMP-2诱导异位骨形成的加速因子，rhBMP-2/磷灰石/胶原/ phosphoryn复合材料可作为骨、牙重建的良好生物材料。少

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。

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。

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

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。

T.Fujii, H.Ariji, F.Kobayashi, T.Saito.: "Bone formation by rhBMP-2 combined with Apatite/Collagen/Phosphophoryn"Bone. 34. S1 (2004)

T.Fujii、H.Ariji、F.Kobayashi、T.Saito.：“rhBMP-2 与磷灰石/胶原蛋白/磷磷蛋白结合形成骨”骨。