The establishment of inducible osteocyte ablation mouse model to elucidate the mechanisms of maxillo-mandibular dysmorphia.

建立诱导性骨细胞消融小鼠模型，阐明上下颌畸形的机制。

• 批准号: 15390641
• 负责人: HOSOYA Akihiro
• 金额: $ 9.28万
• 依托单位: Matsumoto Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15390641/
• 关键词: osteocyte; mechanical stress; apotosis; bone remodeling; estrogen receptor; tamoxifen; Fas; osteoblast; 機械的刺激; プロスタグランジンE_2; RANKL; PGE_2受容体; RAW264.7; NF-κB; p38 MAPK; 破骨細胞

In maxillo-mandibular growth and development, osteocytes absorb mechanical stress generated by muscle contraction and deform bone structure. Thus, osteocytes are thought to be mechanosensory cells that respond to mechanical stress by sending signals to other bone cells to initiate bone remodeling. However, it has been difficult to show that osteocytic lineage are involved in bone remodeling in vivo. In present study, to define the role of osteocytes in the control of bone remodeling, we have tried to generate an inducible osteocyte ablation mouse model. Precisely, transgenic mice have been generated with mouse dentin matrix protein-1 (dmp-l) promoter, which is used to drive expression of Fas fused to mutant estrogen receptor (Fas-ER^t). Dmp-1 is an osteocyte-specific gene, and is expressed only in differentiated osteocytes but not in osteoblasts. Cells expressing (Fas-ER^t) die of tamoxifen treatment, expression of Fas-ER^t in osteocytes should allow inducible ablation of differentiated osteocytes in vivo without affecting the precursor cells, osteoblasts. First, we generated fusion genes composed of various length of the mouse dmp-1promoter fused to GFP reporter gene. The dmp-1-GFP fusion genes were transfected into osteoblastic MC3T3-E1 cells and some stable transformants were obtained. The transformants were cultured for 30 days and GFP activity was measured every days. Osteocyte-specific enhancer activity was observed in region between -1200 and -800bp. Next, we constructed Flag-tagged Fas-ER^t fusion gene and transiently transfected the gene into MC3T3-E1 cells. We tried to induce apoptosis in Flag-Fas-ER^t expressing cells by addition of anti-Flag antibody and tamoxifen, but we were able to induce cell death in only 30% population of these cells. We have examined another system that is more effective than Fas-ER^t. We have considered to use diphtheria-toxin receptor or herpes simplex virus thymidine kinase for inducible cell ablation system.

在上下颌骨生长发育过程中，骨细胞吸收肌肉收缩产生的机械应力，使骨结构变形。因此，骨细胞被认为是机械感觉细胞，通过向其他骨细胞发送信号来响应机械应力，从而启动骨重塑。然而，很难证明骨细胞谱系在体内参与骨重塑。在本研究中，为了明确骨细胞在骨重塑控制中的作用，我们试图建立一个诱导骨细胞消融小鼠模型。准确地说，转基因小鼠已经产生了小鼠牙本质基质蛋白-1 （dmp- 1）启动子，该启动子用于驱动Fas融合到突变雌激素受体（Fas- er ^t）的表达。Dmp-1是一种骨细胞特异性基因，仅在分化的骨细胞中表达，而在成骨细胞中不表达。表达Fas-ER^t的细胞在他莫昔芬治疗后死亡，骨细胞中Fas-ER^t的表达应该允许在体内诱导消融分化的骨细胞而不影响前体细胞成骨细胞。首先，我们将不同长度的小鼠dmp-1启动子与GFP报告基因融合生成融合基因。将bmp -1- gfp融合基因转染成骨细胞MC3T3-E1，获得稳定的转化体。转化体培养30天，每天检测GFP活性。在-1200和-800bp之间的区域观察到骨细胞特异性增强子活性。接下来，我们构建了flag标记的Fas-ER^t融合基因，并将其瞬时转染到MC3T3-E1细胞中。我们试图通过添加抗flag抗体和他莫昔芬来诱导表达Flag-Fas-ER^t的细胞凋亡，但我们只能诱导30%的细胞死亡。我们研究了另一个比Fas-ER^t更有效的系统。我们考虑用白喉毒素受体或单纯疱疹病毒胸苷激酶作为诱导细胞消融系统。

项目成果

Experimental spinal fusion with recombinant human bone morphogenetic Protein-2 delivered by a synthetic polymer and beta-tricalcium phosphate in a rabbit model.

在兔模型中使用合成聚合物和 β-磷酸三钙传递的重组人骨形态发生蛋白 2 进行实验性脊柱融合。

• 发表时间: 2005
• 期刊: Spine 30
• 作者: Namikawa T;Terai H;Suzuki E;Nakamura H;Takaoka K.
• 通讯作者: Takaoka K.

Itoh K.et al.: "LPS promotes the survival of osteoclasts via toll-like receptor 4, but cytokine production of osteoclasts in response to LPS is different from that of macrophages."Journal of Immunology. 170・7. 3688-3695 (2003)

Itoh K.等人：“LPS通过Toll样受体4促进破骨细胞的存活，但破骨细胞响应LPS的细胞因子产生与巨噬细胞不同。”免疫学杂志170・7。 2003）

MyD88 but not TRIF is essential for osteoclastogenes is induced by lipopolysaccharide, diacyl lipopeptide, and IL-1α.

MyD88 而不是 TRIF 对于脂多糖、二酰基脂肽和 IL-1α 诱导的破骨细胞至关重要。

• 发表时间: 2004
• 期刊: Journal of Experimental Medicine 200(5)
• 作者: Sato N;Takahashi N;Suda K;Nakamura M;Yamaki M;Ninomiya T;Kobayashi Y;Takada H;Shibata K;Yamamoto M;Takeda K;Akira S;Noguchi T;Udagawa N.
• 通讯作者: Udagawa N.

Suda K.et al.: "Suppression of osteoprotegerin expression by prostaglandin E_2 is crucially involved in LPS-induced osteoclast formation."Journal of Immunology. 172・4. 2504-2510 (2004)

Suda K.等人：“前列腺素E_2对骨保护素表达的抑制对于LPS诱导的破骨细胞形成至关重要。”免疫学杂志172·4（2004）。

Muramyl dipeptide enhances osteoclast formation induced by lipopolysaccharide, IL-1α and TNFα through Nod2-mediated signaling in osteoblasts.

Muramyl 二肽通过 Nod2 介导的成骨细胞信号传导增强脂多糖、IL-1α 和 TNFα 诱导的破骨细胞形成。

• 发表时间: 2005
• 期刊: J Immunol (in press)
• 作者: Mizoguchi T et al.;Kobayashi Y et al.;Kobayashi Y et al.;Yang S et al.
• 通讯作者: Yang S et al.