Analysis of function of DMP-1 during reparative dentinogenesis and its application for dentin regenerative therapy

DMP-1在牙本质修复过程中的功能分析及其在牙本质再生治疗中的应用

• 批准号: 17591989
• 负责人: NOGUCHI Nobuo
• 金额: $ 2.18万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591989/
• 关键词: DMP-1; Osteopontin; Osteocalcin; Odontoblast; Reparative Dentin; Regeneration of Dentin; osteopontin

To clarify the functional role of DMP-1 during reparative dentinogenesis, the following three experiments were performed.1. The expression of Osteopontin (OPN) and Osteocalcin (OCN) was evaluated after cavity preparation on rat molars using immunohistochemistry. OPN was expressed 7 days after cavity preparation along the dentinal tubules beneath the cavity, and continuously observed until day 28. OCN was detected not only beneath the cavity prepared but also in whole dentin from day 0 to 28.2. We examined the chronological gene expression of DMP-1 and OPN in the pulpal cells was by real-time RT-PCR methods. The tooth was extracted and total RNA was extracted from the pulpal cells after cavity preparation, and then mRNA expression of DMP-1 and OPN was quantitatively measured using comparative Ct methods. The mRNA expression of DMP-1 and OPN was found to be increased in advance the immunohistochemical appearance of proteins.Increased mRNA expression of DMP-1 and OPN was observed in reaction to the stimulation to the pulp by cavity preparation, and DMP-1 and OPN were found to be secreted following expression of mRNA of these molecules in the process of reparative dentin formation. These findings suggest that DMP-1 and OPN may play some important functional role in the healing process of pulp tissues.3. We attempted to produce recombinant DMP-1. After GST-fusion protein was created in Escherichia coli expression construct vector, recombinant DMP-1 was purified by affinity chromatography on GST beads. As it proved that purified DMP-1 was degraded and fragmented using SDS-PAGE, purification condition was modified and we successfully obtained the recombinant DMP-1.

为了阐明在修复性牙本质形成过程中，miR-1的功能作用，进行了以下三个实验。采用免疫组织化学方法检测大鼠磨牙窝洞预备后骨桥蛋白（OPN）和骨钙素（OCN）的表达。窝洞制备后沿着牙本质小管表达OPN，并持续观察至第28天。OCN不仅在洞内被检测到，而且从0到28.2天在整个牙本质中被检测到。采用实时荧光定量RT-PCR方法检测牙髓细胞中骨桥蛋白（OPN）和骨桥蛋白（OPN）基因的时序性表达。取患牙，制备窝洞后，提取牙髓细胞总RNA，采用对比Ct法定量检测牙髓组织中骨桥蛋白（OPN）和骨桥蛋白（OPN）mRNA的表达。结果发现，牙髓组织中OPN和OPN的mRNA表达在蛋白免疫组化出现之前就已增加，牙髓组织中OPN和OPN的mRNA表达在洞制备刺激牙髓时增加，在修复性牙本质形成过程中，OPN和OPN的mRNA表达与牙髓组织中OPN和OPN的mRNA表达同步增加。这些结果表明，牙髓组织中的OPN和β-淀粉样蛋白-1在牙髓组织的愈合过程中可能发挥着重要的功能作用.我们试图生产重组的p-l。在大肠杆菌表达载体中构建GST融合蛋白后，通过GST珠亲和层析纯化重组GST-1。经SDS-PAGE电泳证实，纯化的重组大肠杆菌EST-I被降解并断裂，因此我们对纯化条件进行了改进，成功地获得了重组大肠杆菌EST-I。

项目成果

Gene expression of DMP1 and Osteopontin during reparative dentinogenesis

修复牙本质发生过程中 DMP1 和骨桥蛋白的基因表达

• 发表时间: 2006
• 期刊: Journal of Dental Research 85(Special Issue B)
• 作者: Y.Takahashi;T.Kawahara;S.Toyosawa;S.Imazato;M.Itoh;S.Tanaka;W.Kiba;N.Noguchi;S.Ebisu
• 通讯作者: S.Ebisu