1992 Fiscal Year Final Research Report Summary
Development of the primary culture system for ameloblasts and establish of their cell lines.
| Project/Area Number |
02557069
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Morphological basic dentistry
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| Research Institution | Osaka University (1991-1992)
Kyushu University (1990) |
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Principal Investigator |
KURISU Kojiro Osaka Univ. Oral Anatomy, Professor, 歯学部, 教授 (50028346)
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| Co-Investigator(Kenkyū-buntansha) |
TABATA Makoto Osaka Univ. Oral Anatomy I , Assistant, 歯学部, 助手 (20243248)
KUKITA Akiko Sage Medical Coll. Microbiology, Assistant, 医学部, 助手 (30153266)
MATSUHASHI Sachiko Saga Medical Coll. Biochemistry, Assistant, 医学部, 助手 (00128141)
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| Project Period (FY) |
1990 – 1992
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| Keywords | ameloblast / primary culture / cell line / tooth germ / transformation / rat / development / oncogene |
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| Research Abstract |
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
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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
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