权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Development of the primary culture system for ameloblasts and establish of their cell lines.

成釉细胞原代培养系统的开发及其细胞系的建立。

基本信息

批准号：
02557069
负责人：
KURISU Kojiro
金额：
$ 8.26万
依托单位：
Osaka University (1991-1992)Kyushu University (1990)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02557069/
关键词：
ameloblast primary culture cell line tooth germ transformation rat development oncogene 発癌遺伝子

项目摘要

Ameloblasts are derived from epithelial cells and are responsible for enamel formation. They secrete enamel matrix components in which amelogenins are the major proteins, the biochemical properties of which are well known. However, little is known about the characteristics of ameloblasts themselves or about the functions of amelogenins. In this study, we developed a novel primary and secondary culture system for ameloblasts using a monoclonal antibody En3 which recognized amelogenin. The cell layer of ameloblasts or preameloblasts in tooth germs of raf mandibular incisors were isolated and cultured in serum-free MCDB 153 medium containing insulin, phosphoethanolamine, ethanolamine, hydrocortisone and bovine pituitary extract. Primary culture was performed on collagen-coated culture plates. The number of cells did not increase for first 3-5 days and then increased gradually. The calcium concentration did not affect cell growth. The cultures consisted of three types of cells; (1) adheren … More t polygonal cells, (2) nonadherent round cells and (3) adherent spindle-shaped cells. When the calcium concentration was shifted from low (0.1mM Ca^<2+>) to high (1mM Ca^<2+>), the number of first type of cell decreased whereas that of third type increased. Immunohistochemical examination with En3 demonstrated that first type of cells were negative whereas other two types were positive. In the secondary culture, cells grew more rapidly on collagen-coated plate than on uncoated ones, but the intensity of the staining with En3 was weaker in the former than in the latter. These results indicate that our culture system enable to culture ameloblasts in differentiation state. Amelogenin content in culture medium was detected by ELISA procedure using En3, indicating that this procedure is useful for the quantitative analysis of the effect of various factors on the ameloblasts.Transformation of ameloblasts were attempted by transfection with SV40 gene by using Lipofectin. Cells in secondary culture were treated by Lipofectin containing SV40. At present time, no cell line of ameloblasts has been obtained. Further trials have been undertaken to obtain the cell lines after the reevaluation of experimental conditions. Less

成釉细胞来源于上皮细胞，负责釉质形成。它们分泌釉基质成分，其中釉原蛋白是主要的蛋白质，其生化特性是众所周知的。然而，对成釉细胞本身的特性以及釉原蛋白的功能知之甚少。在这项研究中，我们开发了一种新的原代和二次培养系统的成釉细胞使用单克隆抗体En3识别釉原蛋白。分离大鼠下切牙牙胚中的成釉细胞或成釉细胞层，并在含有胰岛素、磷酸乙醇胺、乙醇胺、氢化可的松和牛垂体提取物的无血清MCDB 153培养基中培养。在胶原包被的培养板上进行原代培养。细胞数在前3 - 5天不增加，然后逐渐增加。钙浓度不影响细胞生长。培养物由三种类型的细胞组成：（1）粘附细胞， ...更多信息 t多边形细胞，（2）非贴壁圆形细胞和（3）贴壁梭形细胞。当钙离子浓度由低（0.1mMCa ^<2 +>）向高（1mMCa ^<2 +>）变化时，第一类细胞数量减少，而第三类细胞数量增加。免疫组化En3染色显示第一型细胞为阴性，而其它两型细胞为阳性。在传代培养中，细胞在胶原包被的平板上比在未包被的平板上生长更快，但前者的En3染色强度比后者弱。这些结果表明，我们的培养体系能够培养处于分化状态的成釉细胞。采用酶联免疫吸附试验（ELISA）检测培养液中釉原蛋白的含量，定量分析各种因素对成釉细胞的影响。用含SV40的Lipofectin处理传代培养的细胞。目前尚未获得成釉细胞系。在重新评估实验条件后，进行了进一步的试验以获得细胞系。少