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

采用RT-PCR方法研究了大鼠下颌磨牙牙胚中肝细胞生长因子（HGF）及其受体c-Met的基因表达规律。原位杂交表明HGF基因在体内仅在牙胚的牙乳头细胞中表达。利用体内免疫组织化学研究揭示了HGF和c-Met在生殖发育过程中的时空分布特征。为了证明HGF在牙齿发育中的功能作用，我们在体外进行了反义硫代寡核苷酸（ODN）阻滞H翻译的实验。在对照实验中，来自胚胎14天的小鼠的牙胚外植体在一种改良的Trowell系统中，在无血清和化学规定的条件下培养两周。其他外植体用15mer反义ODN或正ODN靶向mRNA培养。在体内观察，对照组和感官处理的外植体组织结构正常。另一方面，反感觉处理的外植体表现出异常的结构，牙釉质器官被一层薄薄的牙本质和牙乳头包围，与对照组和感觉处理的外植体相比，呈现“内向外”的结构，尽管成釉细胞和成牙细胞的细胞分化并未受到抑制。经重组人-反义ODN处理的外植体发育正常，说明外源HGF使外植体从反义ODN引起的结构异常中恢复过来。BrdU掺入实验结果提示，HGF翻译阻滞导致牙内牙釉质上皮和牙乳头增殖活性失衡，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.