Cloning of the Genes Specifically Expressed in Teeth

我们构建了牛牙周韧带（PDL）的cDNA文库，并尝试用基因减法技术克隆牛牙周韧带中不表达的基因，但实验失败。PDL是一种独特的组织，它在咬合引起的一定程度的机械应力下仍能保持其功能。我们推测PDL细胞中的Ca信号和细胞骨架元件在这种独特性中发挥了重要作用。由于S100A4是一种钙结合蛋白，并与细胞骨架元件相互作用，因此我们重点研究了该蛋白在PDL中的作用。从PDL cDNA文库中克隆牛S100A4 cDNA。口腔组织中S100A4 mRNA表达水平最高。该基因在萌牙PDL中的表达高于未萌牙的OPDL，在培养的PDL细胞中施加机械应力可提高该基因的表达水平，说明机械应力对S100A4的表达有促进作用。免疫组织学研究表明，S100A4在PDL中的细胞间和细胞外定位。PDL细胞培养基的Western blotting和S^<35>-蛋氨酸标记的PDL细胞培养液分析表明，培养基中存在S100A4。因此，PDL细胞可能在细胞外分泌S100A4。成骨细胞在培养中产生矿化组织，在该培养体系中加入重组S100A4蛋白可抑制矿化。在颅骨发育过程中，早期可以检测到S100A4 mRNA的表达，但在矿化开始时无法检测到其表达。原位杂交实验未检测到成骨细胞中S100A4的表达。因此，S100A4可能是矿化的抑制因子。我们可以得出结论，机械应力刺激PDL细胞中S100A4的表达，PDL细胞分泌S100A4作为矿化抑制剂，但还需要进一步的研究来证明这一点。

We constructed cDNA library of bovine periodontal ligament (PDL) and then tried to clone cDNA of the genes specifically expressed in PDL not expressed in calvaria, using the gene subtraction technique, however this experiment was unsuccessful. PDL is a unique tissue because it maintains its function under extent mechanical stress caused by occlusion. We speculate that Ca signaling and cytoskeletal elements in PDL cells play important roles in this uniqueness. Since S100A4 is a Ca-binding protein and interacts with cytoskeletal elements, we focused our research on this protein in PDL.We cloned bovine S100A4 cDNA from PDL cDNA library. The highest level of S100A4 mRNA expression was ovserved among the oral tissues examined. PDL of erupted teeth expressed this gene higher than OPDL of unerupted teeth and application of mechanical stress to cultured PDL cells increased the expression level of this gene, indicating stimulative effect of mechanical stress on S100A4 expression. Immunohistological study demonstrated intercellular and extracellular localization of S100A4 in PDL.Western blotting of the culture medium of PDL cells and analysis of S^<35>-methionine labeled culture of PDL cells demonstrated the existence of S100A4 in the medium. Thus, it is likely that PDL cells secrete S100A4 extracellularly. Osteogenic cells produce mineralized-tissue in culture and addition of recombinant S100A4 protein in this culture system inhibited mineralization. In calvaria development, S100A4 mRNA expression was detected in early stage, however its expression was undetectable when mineralization started. Furthermore, S100A4 expression of osteoblasts was not detected in situ Hybridization experiment. Thus, it is possible that S100A4 is a inhibitory factor for mineralization. We can conclude that mechanical stress stimulates S100A4 expression in PDL cells and PDL cells secrete S100A4, which acts as an inhibitor for mineralization although further study is necessary to prove this story.