Search for the bioactive molecules implicated in the regulation of human periodontal ligament cell functions

寻找参与调节人牙周膜细胞功能的生物活性分子

• 批准号: 09470464
• 负责人: SHIRAKAWA Tetsuo
• 金额: $ 3.39万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470464/
• 关键词: nitric oxide (NO); nitric oxide synthase (NOS); endothelial NOS; periodontal ligament cell; cyclic tension force; RANKL; osteoprotegerin; ヒト歯根膜由来細胞; RT-PCR; チロシンキナーゼ; Embryonal Fyn-associated substrate; ヒト歯根膜細胞; 細胞成長因子; サイトカイン; 三又神経; ニューロン

The periodontal ligament (PDL) has a critical role of keeping the tooth root intact. We found that cultured human PDL cells (hPDL cells) spontaneously produced nitric oxide (NO). This NO production was not affected by culturing the cells with interleukin-1 β(IL-1 β), However, the NO production was enhanced by stimulating the cells with cyclic tension forces. In an unstimulated hPDL cell culture, concentration of NOィイD22-ィエD2 and NOィイD23-ィエD2 (NOィイD22-ィエD2/NOィイD23-ィエD2), the oxidized products of NO, increased to 140% of the initial value during the first 12 h in newly exchanged medium. In contrast, NOィイD22-ィエD2/NOィイD23-ィエD2 showed a 3-fold increase when the cells had been subjected to cyclic tension forces for 12 h.We employed RT-PCR method for the detection of NO synthase mRNA in the Hpdl cells. Endothelial NO synthase (ecNOS) mRNA was expressed in both stimulated and unstimulated Hpdl cells whereas inducible NO synthase (iNOS) mRNA was detected in neither culturing condition. Furthermore, we found strong ecNOS but not iNOS immunoreactivity in the stimulated Hpdl cells. Mechanically stimulated Hpdl cells were suggested to modulate the functions of periodontal tissue by the upregulated NO production.Expression of receptor activator of NF-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG) was investigated in cultures of hPDL cells. Both RANKL and OPG mRNAs were expressed in the hPDL cells and Northern blot analysis revealed that the OPG mRNA expression was down-regulated remarkably by an application of 1 alpha, 25(OH)ィイD22ィエD2 vitamin D3 (1,25-(OH)ィイD22ィエD2DィイD23ィエD2) and dexamethasone (Dex). In contrast, RANKL mRNA expression was up-regulated by the same treatment. Tartrate-resistant acid phosphatase-positive multinuclear cells were markedly induced when the hPDL cells were co-cultured with bone marrow cells in the presence of an anti-OPG antibody together with 1,25-(OH)ィイD22ィエD2DィイD23ィエD2 and Dex.

牙周膜（PDL）在保持牙根完整方面具有关键作用。我们发现培养的人PDL细胞（hPDL细胞）自发产生一氧化氮（NO）。白细胞介素1 β（IL-1 β）对细胞NO的产生无影响，而周期性张力刺激可促进细胞NO的产生。在未受刺激的hPDL细胞培养中，NO的氧化产物NO还原酶D22-还原酶D2和NO还原酶D23-还原酶D2（NO还原酶D22-还原酶D2/NO还原酶D23-还原酶D2）的浓度在新更换的培养基中的前12 h内增加到初始值的140%。相反，当细胞经受循环张力12 h时，NO合成酶D22-D23-D23/D23-D23显示3倍的增加。我们采用RT-PCR方法检测Hpdl细胞中NO合成酶mRNA。内皮型一氧化氮合酶（ecNOS）mRNA在刺激和未刺激的Hpdl细胞中表达，而诱导型一氧化氮合酶（iNOS）mRNA在两种培养条件下均未检测到。此外，我们发现强烈的ecNOS，但不是诱导型一氧化氮合酶的免疫反应在刺激Hpdl细胞。机械刺激HPDL细胞后，NO的产生增加，从而调节牙周组织的功能。本研究观察了HPDL细胞中NF-κ B B配体受体激活因子（RANKL）及其诱饵受体骨保护素（OPG）的表达。RANKL和OPG mRNA在hPDL细胞中均有表达，北方印迹分析显示，1 α，25-（OH）β D22 β D2维生素D3（1，25-（OH）β D22 β D23 β D2）和地塞米松（Dex）可显著下调OPG mRNA的表达。与此相反，RANKL mRNA表达上调相同的处理。当在抗OPG抗体与1，25-（OH）β-D22 β-D2 D β-D23 β-D2和Dex存在下将hPDL细胞与骨髓细胞共培养时，显著诱导了抗酒石酸酸性磷酸酶阳性多核细胞。

项目成果

Kikuiri, T., Hasegawa, T., Yoshimura, Y., Shirakawa, T. and Oguchi, H.: "Cyclic tension force activates nitric oxide production in cultured human periodontal ligament cells"Journal of Periodontology. vol.71. 533-539 (2000)

Kikuiri, T.、Hasekawa, T.、Yoshimura, Y.、Shirakawa, T. 和 Oguchi, H.：“循环张力激活培养的人牙周膜细胞中一氧化氮的产生”《牙周病学杂志》。

菊入崇,白川哲夫,長谷川智一,吉村善隆,竹山禎章,加我正行,小口春久: "ヒト歯根膜由来細胞におけるNO産生について"小児歯科学雑誌. 37. 768-774 (1999)

Takashi Kikuiri、Tetsuo Shirakawa、Tomokazu Hasekawa、Yoshitaka Yoshimura、Yoshiaki Takeyama、Masayuki Kaga、Haruhisa Oguchi：“关于人类牙周膜衍生细胞中的 NO 产生”《儿童牙科杂志》37. 768-774 (1999)。

菊入 崇、白川哲夫、長谷川智一、吉村善隆ほか3名: "ヒト歯根膜由来細胞におけるNO産生について"小児歯科学雑誌. 37.4. 768-774 (1999)

Takashi Kikuiri、Tetsuo Shirakawa、Tomokazu Hasekawa、Yoshitaka Yoshimura 等 3 人：“关于人类牙周膜衍生细胞中的 NO 产生”《儿科牙科杂志》37.4（1999 年）。