Analysis of the function of hepatocyte growth factor in the development of tooth.

应用RT-PCR技术研究了肝细胞生长因子(HGF)及其受体c-Met基因在大鼠下颌磨牙牙胚中的表达模式。原位杂交结果表明，HGF基因仅在活体牙胚的牙乳头细胞中表达。免疫组织化学研究揭示了HGF和c-Met在胚胎发育过程中的时空分布特征。为了证实HGF在牙齿发育中的作用，体外用反义硫代寡核苷酸(ODN)抑制H翻译。在对照实验中，胚胎14日龄小鼠的牙胚外植体在改良的Trowell系统中，在无血清和化学条件下培养两周。用15聚体的反义或正义寡核苷酸对其他外植体进行培养，对照和正义处理的外植体在体内均显示出正常的组织结构。另一方面，反义处理的外植体显示出一种异常的结构，釉质器官被一层薄薄的牙本质和牙乳头包围，与对照和正义处理的外植体相比，看起来是由内向外的，尽管成釉细胞和成牙本质细胞的细胞分化没有受到抑制。重组人联合反义ODN处理的外植体发育正常，说明外源性HGF将外植体从反义ODN诱导的异常结构中解救出来。BrdU掺入实验结果表明，HGF翻译停滞导致内釉质上皮和牙乳头的增殖活性失衡，导致牙胚结构异常。这些结果表明，HGF参与了小鼠磨牙的形态发生。

The pattern of gene expression for hepatocyte growth factor (HGF) and its receptor, c-Met were revealed in the tooth germ of rat mandibular molars using RT-PCR.In situ hybridization demonstrated that the HGF gene was expressed only in the cells of the dental papilla of the tooth germ in vivo. The characteristic temporospatial distribution of HGF and c-Met during germ development was revealed using immunohistochemical studies in vivo. In order to demonstrate the functional role playd by HGF in tooth development, H translation arrest by antisense phosphorothioate oligonucleotide (ODN) was carried out in vitro. In the control experiment, explants of tooth germs from embryonic 14 day mice were cultured in a modification of Trowell's system under serum-free and chemically defined conditions for two weeks. Other explants were cultured with 15mer antisense or sense ODN targeted to the mRNA.Both the control and the sense-treated explants showed normal histological structure, as observed in vivo. On the other hands, anti-sense-treated explants exhibited an abnormal structure in which the enamel organs were surrounded by a thin layr of dentin and dental papilla, appearing 'inside-out' compared to the control and sense-treated explants, although the cytodifferentiation of ameloblasts and odontoblasts was not inhibited. The explants treated with recombinant human combined with antisense ODN showed normal deve-lopment, indicating the exogenous HGF rescued the explants from the abnormal strcture caused by antisense ODN.The findings of a BrdU incorporation experiment suggest that the imbalance between the proliferation activity of the inner enamel epithelium and that of the dental papilla caused by HGF translation arrest results in the abnormal structure of the tooth germ. These results indicate that HGF is involved in the morphogenesis of the murine molar.