Effects of Melatonin on regenerative treatment in periodontal disease

(1)在龈沟液中检测到前列腺素E_2(PGE_2)，它是一种强有力的炎症介质。本研究旨在探讨前列腺素E_2(PGE_2)对脂多糖(LPS)诱导的人牙龈成纤维细胞(HGFs)产生IL-8的调节作用。HGFs取自临床上无牙周病的健康供者。将细胞悬浮在添加5%热灭活FCS的OMEM中，以1×104个/孔的细胞密度接种于96孔板中，在37℃5%CO_2中孵育3天。实验前24小时取下培养液，用含1%胎牛血清的DMEM代替。然后将HGFs暴露于牙龈卟啉单胞菌、伴生放线杆菌或大肠杆菌的不同浓度的内毒素中。在加入脂多糖制剂前，将不同浓度的PGE2作用于HGFs培养2 h，观察PGE2对HGFs生长的影响。培养结束后，收集培养上清，检测IL-8u…更多的人唱着酶联免疫试剂盒。逆转录-聚合酶链式反应(RT-PCR)检测PGE2刺激后IL-8mRNA的表达。HGFs产生IL-8与孵育时间和内毒素剂量有关。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。用PGE2预先刺激HGFs，然后再暴露于LPS，IL-8的产生是未处理细胞的两到三倍。除草剂A可完全阻断IL-8的分泌。这些结果表明，PGE2可以调节HGFs分泌细胞因子，增加细胞在脂多糖作用下产生的IL-8。脂多糖诱导的IL-8的产生依赖于酪氨酸磷酸化途径。(2)目的：探讨牙本质中主要的非胶原蛋白磷酸化蛋白在rhBMP-2诱导体内硬组织形成过程中是否起到了rhBMP-2的辅助作用。方法：以吸附在多孔羟基磷灰石上的牙胶原为载体，诱导异位成骨。在磷灰石/胶原复合材料中加入5微克的重组人骨形态发生蛋白-2和1毫克的磷酸盐。4周龄雄性Wistar大鼠背部双侧皮下植入rhBMP-2/磷灰石/胶原蛋白/磷酸化磷脂。同时植入rhBMP-21磷灰石/胶原或rhBMP-2/磷灰石作为对照。植入后每周间隔3周处死大鼠，摘除种植体。对植入物进行了组织学和生化分析。结果：种植后1周(1周)，所有种植体均可见部分小部位成骨。2周时，rhBMP-2复合磷灰石/胶原蛋白/磷灰石的成骨作用明显多于复合磷灰石/胶原蛋白，3周时种植体表面已有骨形成，并发生骨改建，骨髓细胞取代了中间的羟基磷灰石颗粒。3周时，rhBMP-21磷灰石/胶原/磷酸磷灰石的ALPase活性约为rhBMP-2/磷灰石/胶原蛋白的2倍。结论：在异位骨形成过程中，磷酸磷灰石起到了促进rhBMP-2诱导异位成骨的作用，提示rhBMP-2/磷灰石/胶原/磷蛋白复合材料有望成为一种良好的骨和牙齿重建生物材料。较少

(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