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; 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

APERT综合征（AS）是由成纤维细胞生长因子受体（FGFR）2引起的错义突变引起的常染色体显性遗传疾病，其特征在于颅内突发发生，外部表面缺乏症，中间缺陷和对称性综合症的临床特征。据报道，FGFR2的配体依赖性构型激活在AS的发病机理中起着结构的作用，但是确切的机制仍有待阐明。经常需要进行手术程序来治疗AS，即急切期待的非侵入性程序的发展。目标：确定AS中颅突的病因机制，并验证与多糖纳米凝胶体外复合的FGFR2S252W（SFGFR2S252W）纯化的固体形式的治疗作用。方法：重组SFGFR2S252W通过亲和色谱纯化，并与纳米凝胶复合。获得了颅骨组织，获得了更多的ROM FGFR2+/S252WMICE（作为小鼠）和野生型小鼠（对照小鼠），并在SFGFR2S252W/纳米凝胶复合物或冠状成功的两侧培养4天。建立了过表达FGFR2IIIC252W（MC3T3-E1-AP）的MC3T3-E1细胞。通过实时PCR或半定量RT-PCR分析MC3T3-E1细胞中的mRNA表达，并通过蛋白质印迹分析蛋白质表达和磷酸化。结果：与对照小鼠中观察到的不同，AS小鼠暴露的冠状缝合增加了Runx2和骨桥蛋白mRNA的表达以及ERK和MEK的加速磷酸化。在AS小鼠的冠状成功中观察到FGF10和FGFR2IIIB的异位表达可能是表皮发育所不可或缺的，而在AS和对照小鼠中都检测到FGFR2IIIC的表达。施用SFGFR2S252W抑制FGF2刺激的增殖； ERK，P38，MEK，SAPK/JNK和AKT的磷酸化；以及MC3T3-E1-AP在体外的矿化。 SFGFR2S252W/纳米凝胶复合物保持AS小鼠冠状成功的通畅性（n = 4/4）；然而，观察到在器官培养中仅应用纳米凝胶（n = 4/4）的侧面发生冲突。结论：FGF10和FGFR2IIIB的异位表达可能诱导AS中的颅突发作，并且适当的纯化SFGFR2S252W的递送可能是治疗AS的有效方法。较少的