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

Research Project

Project/Area Number 02557069
Research Category

Grant-in-Aid for Developmental Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field Morphological basic dentistry
Research InstitutionOsaka University (1991-1992)
Kyushu University (1990)

Principal Investigator

KURISU Kojiro  Osaka Univ. Oral Anatomy, Professor, 歯学部, 教授 (50028346)

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)
上野 和則  日本歯科薬品, 研究開発部, 部長
Project Period (FY) 1990 – 1992
Project Status Completed (Fiscal Year 1992)
Budget Amount *help
¥12,900,000 (Direct Cost: ¥12,900,000)
Fiscal Year 1992: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1991: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥10,200,000 (Direct Cost: ¥10,200,000)
Keywordsameloblast / primary culture / cell line / tooth germ / transformation / rat / development / oncogene / 発癌遺伝子
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 … 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

Report

(4 results)
  • 1992 Annual Research Report   Final Research Report Summary
  • 1991 Annual Research Report
  • 1990 Annual Research Report
  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] T.INAI: "Demonstration of amelogenin in the enamel-free area in the occulusal cusps of rat molar tooth germs:Immunofluorescent and immunoelectron microscopic studies." Anatomical Record. 233. 588-596 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] K,NAGATA: "Demonstration of type III collagen in dentin of mouse molars." Matrix. 12. 448-455 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] K,KUKITA: "Primary and secondary culture of rat ameloblast in serum-free medium." Calcified Tissue International. 51. 393-398 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] T.INAI: "Immunohistochemical detection of an enamel-related epitope in rat bone at early stage of osteogenesis." Histochemistry.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Inai, T.: "Demonstration of amelogenin in the enamel-free area in the occlusal cusps of rat molar tooth germs: Immunofluorescent and immunoelectron microscopic studies." Ant. Rec.233. 588-596 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Nagata, K.: "Demonstration of type III collagen in dentin of mouse molars." Matrix. 12. 448-455 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Kukita, A.: "Primary and secondary culture of rat ameloblast in serum-free medium." Calcif. Tissue Intn.51. 393-398 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Inai, T.: "Immunohistochemical detection of an enamel-related epitope in rat bone at early stage of osteogenesis" Histochemistry.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] T.Inai: "Demonstration of amelogenin in the enamel-free area in the occlusal cusps of rat molar tooth germs:Immunofluorescent and immunoelectron microscopic studies." Anatomical Record. 233. 588-596 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] K.Nagata: "Demonstration of type III collagen in dentin of mouse molars." Matrix. 12. 448-455 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] K.Kukita: "Primary and secondary culture of rat ameloblast in serum-free medium." Calcified Tissue Intermational. 51. 393-398 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] T.Inai: "Immunohistochemical detection of an enamel-related epitope in rat bone at early stage of osteogenesis." Histochemistry.

    • Related Report
      1992 Annual Research Report
  • [Publications] A.Kukita,H.Harada,T.Kukita,T.Inai,S.Matsuhashi,K.Kurisu: "Primary and secondary culture of rat ameloblasts in serum-free medium." Calcif.Tissue Intn.(1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] K.Nagata,Y.H.Huang,Y.Ohsaki T.,Kukita,K.Nakata,K.Kurisu: "Demonstration of type III collagen in dentin of mouse molars." Matrix,. (1992)

    • Related Report
      1991 Annual Research Report

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Published: 1990-04-01   Modified: 2016-04-21  

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