Non-collagenous proteins in the apical portion of the forming root in porcine permanent incisor tooth germs

研究了猪恒切牙胚成根尖部组织样品中牙釉质蛋白的检测和表达。通过组织化学方法证实，样品中含有牙本质前质、牙本质和薄骨水泥作为细胞外基质，同时含有大量的细胞，如成牙髓细胞、牙乳头细胞、成牙骨质细胞、滤泡细胞和碎片化的HERS细胞。胍溶液中含有大量组蛋白，与抗牙釉质蛋白血清发生交叉反应。由于提取液中含有少量的组蛋白，因此采用醋酸溶液进行免疫印迹研究是合理的。免疫印迹法检测到淀粉原蛋白（Amelogenin）、釉蛋白（enamelin）和鞘蛋白（sheathin），但含量较少。在APFR样品中，其cdna扩增产物也证实了珐琅质蛋白的表达，并可能与Hertwig上皮根鞘断裂的细胞有关。淀粉原蛋白的表达不优于釉质蛋白和鞘蛋白。这与参与釉质基质形成的秘书成釉细胞的表达模式不同。这些结果表明，在APFR中发现的淀粉原蛋白并没有形成由淀粉原蛋白胶束构成的三维结构，这是在秘书釉质基质结构上提出的。牙釉质蛋白衍生物的免疫化学检测显示其存在降解现象。目前还不清楚与这些降解有关的蛋白酶，因为在明胶和酪蛋白酶谱上没有检测到釉质基质丝氨酸蛋白酶1和釉质溶酶的活性。因此，牙釉质蛋白很容易扩散，降解成未来牙骨质的基质。它们的一些衍生物可能在骨质形成中起作用。

Detection and expression of enamel proteins were explored on the tissue sample prepared from the apical portion of forming root (APFR) in porcine permanent incisor tooth germs. It was confirmed according to a histochemical method that the sample contained predentin, dentin and thin cement as extracellular matrices, and also contained a lot of cells as odontoblasts, dental papilla cells, cementoblasts, follicular cells, and fragmentized HERS cells. When a guanidine solution was employed for the extraction, it contained a lot of histons, which were croos-reacted with anti-enamel proteins sera. The employment of acetic acid solution was reasonable for the immunoblot studies, since the extractant contained small amount of histons.Amelogenin, enamelin and sheathlin were detected by immunoblot analyses, but in a small amounts. The expressions of enamel proteins were also proved in the APFR sample with PCR amplification products of their cDNAs, and may be related to the cells of fragmentized Hertwig's epithelial root sheath. The amelogenin was not expressed superior to the enamelin and sheathlin. It was different from the expression pattern of the secretary ameloblasts involved to the enamel matrix formation. These results suggest that the amelogenins found in the APFR do not form the three-dimensional structure constructed by amelogenin micelles, which is proposed on the secretary enamel matrix structure. The immunochemical detections of enamel proteins' derivatives showed the occurrences of their degradation. It is at present unclear about the proteinases related to these degradations, since the activities of enamel matrix serine proteinase 1 and enamelysin were not detected on gelatin and casein zymograms. therefore, the enamel proteins spread easily out with degradation into the matrix of future cementum. Some of their derivatives may play a role in cementum formation.