Identification of the cell group participating in the onset of craniosynostosis and study on the differentiation control

参与颅缝早闭发生的细胞群鉴定及分化调控研究

• 批准号: 23792418
• 负责人: KOBAYASHI Yukiho
• 金额: $ 2.75万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2011
• 资助国家: 日本
• 起止时间: 2011 至 2012
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-23792418/
• 关键词: craniosynostosis; Apert syndrome; osteoblast; 先天性疾患; 歯科矯正学; 頭蓋冠縫合部早期癒合症; FGF; FGFR; Apert症候群; 頭蓋冠縫合部; 先天異常; 骨芽細胞分化

Apert syndrome (AS), an autosomal dominant inherited disorder caused by missense mutation resulting from amino acid substitutions in the fibroblast growth factor receptor (FGFR) 2, is characterized by the major clinical features of craniosynostosis, exophthalmus, midface deficiency, and symmetric syndactyly of the hands and feet. Ligand-dependent constitutive activation of FGFR2 has been reported to play a causative role in the pathogenesis of AS, however the precise mechanism remains to be elucidated. Surgical procedures are frequently required to treat the AS, thedevelopment of non-invasive procedures for treating AS are keenly awaited. Objectives: To determine etiological mechanisms of craniosynostosis in AS, and verify the therapeutic effects of the purified soluble form of FGFR2S252W (sFGFR2S252W) complexed with polysaccharide nanogel in vitro. Methods: Recombinant sFGFR2S252W was purified by affinity chromatography and was complexed with nanogel. Calvarial tissues were obtained f … More rom Fgfr2+/S252Wmice (AS mice) and wild-type mice (control mice), and were cultured for 4 days in the presence of either sFGFR2S252W/nanogel complex or vehicle nanogel on either side of the coronal suture. MC3T3-E1 cells overexpressing FGFR2IIIcS252W (MC3T3-E1-Ap) were established. The mRNA expression in MC3T3-E1 cells was analyzed by real-time PCR or semi-quantitative RT-PCR, and protein expression and phosphorylation were analyzed by Western blotting. Results: The coronal suture of the AS mice exhibited increased Runx2 and Osteopontin mRNA expression, as well as accelerated phosphorylation of ERK and MEK, unlike that observed in the control mice. The ectopic expression of Fgf10 and Fgfr2IIIb, which are probably indispensable for epidermal development, were observed in the coronal suture of the AS mice, whereas Fgfr2IIIc expression was detected in both the AS and the control mice. Administration of sFGFR2S252W inhibited FGF2-stimulated proliferation; phosphorylation of ERK, p38, MEK, SAPK/JNK, and Akt; and the mineralization of MC3T3-E1-Ap in vitro. The sFGFR2S252W/nanogel complex maintained the patency of the coronal sutures in the AS mice (n = 4/4); however, synostosis was observed to occur on the side where only nanogel was applied (n = 4/4) in the organ culture. Conclusion: The ectopic expression of Fgf10 and Fgfr2IIIb probably induce the onset of craniosynostosis in AS, and anappropriate delivery of purified sFGFR2S252W could be an effective method for treating AS. Less

阿佩尔综合征（AS）是一种常染色体显性遗传性疾病，由成纤维细胞生长因子受体（FGFR）2氨基酸取代引起的错义突变引起，其主要临床特征为颅缝早闭、突眼、中面部缺陷和对称性并指。据报道，FGFR2 的配体依赖性组成性激活在 AS 的发病机制中发挥着致病作用，但确切的机制仍有待进一步研究。治疗 AS 经常需要手术治疗，迫切需要开发治疗 AS 的非侵入性手术。 目的：确定 AS 颅缝早闭的病因机制，并验证纯化的可溶性 FGFR2S252W 的治疗效果。 (sFGFR2S252W)与多糖纳米凝胶体外复合方法：通过纯化重组sFGFR2S252W。从 Fgfr2+/S252W 小鼠（AS 小鼠）和野生型小鼠（对照小鼠）中获得颅骨组织，并在 sFGFR2S252W/纳米凝胶复合物或载体存在下培养 4 天。冠状缝两侧的纳米凝胶过度表达 FGFR2IIIcS252W。 (MC3T3-E1-Ap)通过实时PCR或半定量RT-PCR分析MC3T3-E1细胞中的mRNA表达，并通过Western blotting分析蛋白表达和磷酸化。与对照小鼠中观察到的异位表达不同，AS 小鼠表现出 Runx2 和骨桥蛋白 mRNA 表达增加，以及 ERK 和 MEK 磷酸化加速。在 AS 小鼠的冠状缝中观察到 Fgf10 和 Fgfr2IIIb，这可能是表皮发育所必需的，而在 AS 和对照小鼠中均检测到 Fgfr2IIIc 表达，施用 sFGFR2S252W 抑制了 FGF2 刺激的 ERK 增殖。 p38、MEK、SAPK/JNK 和 Akt；以及体外 MC3T3-E1-Ap sFGFR2S252W/纳米凝胶复合物保持 AS 小鼠冠状缝的通畅（n = 4/4）；然而，观察到仅应用纳米凝胶的一侧发生骨融合（n = 4/4）。 4/4) 在器官培养中结论：Fgf10 和 Fgfr2IIIb 的异位表达可能诱导了 Fgf10 和 Fgfr2IIIb 的发生。 AS 颅缝早闭，适当递送纯化的 sFGFR2S252W 可能是治疗 AS 的有效方法。

项目成果

Runx2発現抑制によるGli3Xt-J/Xt-Jマウス頭蓋冠縫合部早期癒合症発症の予防効果

抑制 Runx2 表达对 Gli3Xt-J/Xt-J 小鼠颅骨缝早期骨联的预防作用

• 作者: 北村良平;川元龍夫;宮本順;樺沢勇司;小村健;黒原一人;天笠光雄;森山啓司;小林起穂 ヴァイスティネン ロッタ ライス デビッド 森山啓司
• 通讯作者: 小林起穂 ヴァイスティネン ロッタ ライス デビッド 森山啓司

マウス頭蓋顎顔面領域の発生過程におけるリラクシン受容体遺伝子発現様相の解析Analysis of the expression pattern of Relaxin receptors during mouse craniofacial development

小鼠颅面发育过程中松弛素受体的表达模式分析

• 发表时间: 2012
• 作者: Duarte C;Kobayashi Y;Kawamoto T;Moriyama K
• 通讯作者: Moriyama K

Expression Pattern of Relaxin Receptors During Mouse Craniofacial Development

小鼠颅面发育过程中松弛素受体的表达模式

• 发表时间: 2012
• 作者: Duarte C;Kobayashi Y;Kawamoto T;Moriyama K
• 通讯作者: Moriyama K

Apert syndrome mutant FGFR2 and its soluble form reciprocally alter osteogenesis of primary calvarial osteoblasts

• DOI: 10.1002/jcp.24021
• 发表时间: 2012-09
• 期刊: Journal of Cellular Physiology
• 影响因子: 5.6
• 作者: Hiroyuki Suzuki;N. Suda;Momotoshi Shiga;Yukiho Kobayashi;Masataka Nakamura;S. Iseki;K. Moriyama

Soluble FGFR2 with Apert mutation inhibits osteoblastic differentiation and proliferation

具有 Apert 突变的可溶性 FGFR2 抑制成骨细胞分化和增殖

• 发表时间: 2013
• 作者: Masako YOSHIZAKI ;Yukiho KOBAYASHI;Keiji MORIYAM
• 通讯作者: Keiji MORIYAM