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

成釉细胞来源于上皮细胞，负责釉质的形成。它们分泌釉质基质成分，其中釉原蛋白是主要的蛋白质，其生化特性众所周知。然而，人们对成釉细胞本身的特性或成釉蛋白的功能知之甚少。在这项研究中，我们利用识别成釉蛋白的单抗EN3建立了一种新的成釉细胞原代和传代培养系统。分离RAF下颌切牙牙胚成釉细胞或前成釉细胞，在含有胰岛素、磷乙醇胺、乙醇胺、氢化可的松和牛脑垂体提取物的MCDB-153无血清培养液中进行培养。在包被胶原蛋白的培养板上进行原代培养。细胞数在前3~5d不增加，以后逐渐增加。钙浓度对细胞生长无明显影响。培养物由三种类型的细胞组成：(1)贴壁…多为多角形细胞，(2)非贴壁圆形细胞，(3)贴壁梭形细胞。当钙浓度由低(0.1 mM)向高(1 MM)转变时，第一类细胞数量减少，第三类细胞数量增加。免疫组织化学EN3染色显示，第一类细胞为阴性，其他两类细胞均为阳性。在二次培养中，细胞在胶原涂层平板上的生长速度快于未涂层平板，但EN3染色强度前者弱于后者。这些结果表明，我们的培养体系能够培养分化状态的成釉细胞。以EN3为底物，用ELISA法检测培养上清液中的成釉蛋白含量，为定量分析各种因素对成釉细胞的影响奠定了基础。用含SV40的脂联素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，已经进行了进一步的试验以获得细胞系。较少

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