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Comparative immunohistochemical analyses of the localization of amelogenin domains during a tooth enamel development inlowervertebrates

低等脊椎动物牙釉质发育过程中釉原蛋白结构域定位的比较免疫组织化学分析

基本信息

批准号：
18592013
负责人：
ISHIYAMA Mikio
金额：
$ 2.53万
依托单位：
The Nippon Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

In order to understand the phylogenetic characterization of amelogenin protein in lower vertebrates, cDNA sequences of amelogenin in reptile (iguana) and amphibia (newt) were determined, then immunohistochemical and western blot analyses were performed in them, using antibodies against the peptides consisting the N-terminus, C-terminus and central domain of their amelogenin. These antibodies were termed as follows by the recognizing portions of amelogenin in iguana and newt : IN (iguana N-terminus), IM (iguana central domain), IC (iguana C-terminus), NN (newt N-terminus), NM (newt central domain) and NC (newt C-terminus). ELISA analysis indicated that each antibody had a high specificity for the corresponding peptide. Immunohistochemical analysis was examined by the protein-A-gold method, for the iguana using IN,IM and IC, and for the newt using NN, NM and NC. Consequently, all antibodies, except NC, showed intense and uniform immunoreactivities over the enamel matrix and the secretory granules of the ameloblasts in iguana and newt, respectively. Only NC showed ununiform distribution of reactivities over the enamel matrix in newt. The intense reactions with NC were concentrated on the surface layer of an enamel matrix and decreased gradually toward the middle layer to deep layer. Similar phenomenon was reported by Uchida, et. al.(1991) by immunohistochemical demonstration of amelogenin in the porcine tooth enamel formation using the antibody against the C-terminus of the porcine amelogenin. The present study, therefore, may indicates that the newt has the degraded mechanism of the amelogenin similar to mammalian amelogenesis. Western blot analysis, however, showed the presence of 22 kDa amelogenin in both newt and iguana but the absence of low molecular weight of amelogenins under 20 kDa in them. These data suggest that the amelogenesis in reptile and amphibia were incomplete for the degradation of amelogenin proteins compared with that in mammals.

为了了解低等脊椎动物中牙釉蛋白的系统发育特征，确定了爬行动物（鬣蜥）和两栖动物（蝾螈）中牙釉蛋白的cDNA序列，然后使用针对其牙釉蛋白N端、C端和中央结构域组成的肽的抗体对它们进行免疫组织化学和蛋白质印迹分析。这些抗体根据鬣蜥和蝾螈中牙釉蛋白的识别部分命名如下：IN（鬣蜥 N 末端）、IM（鬣蜥中央结构域）、IC（鬣蜥 C 末端）、NN（蝾螈 N 末端）、NM（蝾螈中央结构域）和 NC（蝾螈 C 末端）。 ELISA分析表明每种抗体对相应的肽段均具有较高的特异性。采用蛋白-A-金法对鬣蜥进行免疫组织化学分析，采用 IN、IM 和 IC，对蝾螈采用 NN、NM 和 NC 进行免疫组织化学分析。因此，除 NC 外，所有抗体分别对鬣蜥和蝾螈的牙釉质基质和成釉细胞的分泌颗粒表现出强烈且均匀的免疫反应性。只有 NC 在蝾螈的牙釉质基质上表现出不均匀的反应性分布。与NC的强烈反应集中在釉质基质的表层，并向中层至深层逐渐减弱。 Uchida 等人也报道了类似的现象。 al.(1991)通过使用抗猪牙釉蛋白C末端的抗体对猪牙釉质形成中的牙釉蛋白进行免疫组织化学论证。因此，本研究可能表明蝾螈具有与哺乳动物釉原形成相似的釉原蛋白降解机制。然而，蛋白质印迹分析显示蝾螈和鬣蜥中均存在 22 kDa 的牙釉蛋白，但不存在低于 20 kDa 的低分子量牙釉蛋白。这些数据表明，与哺乳动物相比，爬行动物和两栖动物的釉原蛋白降解不完全。