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，负责牙釉质的形成。它们分泌珐琅质基质成分，其中淀粉原蛋白是主要蛋白质，其生化特性是众所周知的。然而，对成釉细胞本身的特性和成釉原蛋白的功能了解甚少。本研究利用单克隆抗体En3建立了一种新的成釉细胞初、二次培养体系。在含胰岛素、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培养基中分离培养raf下切牙牙胚成釉细胞或前成釉细胞层。原代培养在胶原包被培养板上进行。细胞数在前3 ~ 5 d不增加，后逐渐增加。钙浓度对细胞生长无影响。培养物包括三种类型的细胞；多为多角形细胞，(2)非贴壁的圆形细胞，(3)贴壁的纺锤形细胞。当钙浓度由低（0.1mM Ca^<2+>）到高（1mM Ca^<2+>）时，第一类细胞数量减少，第三类细胞数量增加。En3免疫组化检查显示第一种细胞为阴性，其他两种细胞为阳性。在二次培养中，细胞在胶原包被板上比在未包被板上生长得更快，但En3染色强度弱于未包被板。这些结果表明我们的培养体系能够培养成釉细胞处于分化状态。采用En3酶联免疫吸附法检测培养基中成釉原蛋白的含量，表明该方法可用于定量分析各种因素对成釉细胞的影响。用Lipofectin转染SV40基因，尝试转化成釉细胞。二次培养细胞用含有SV40的脂质体素处理。目前尚未获得成釉细胞细胞系。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。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