| Project/Area Number |
07044279
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
NAKASHIMA Misako Kyushu University, Research Associate, 歯学部, 助手 (20207773)
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| Co-Investigator(Kenkyū-buntansha) |
NAGASAWA Hisashi Prof.emeriti, Kyushu University, Faculty of Dentistry, 歯学部, 名誉教授 (10013848)
AKAMINE Akifumi Prof., Kyushu University, Faculty of Dentistry, 歯学部, 教授 (00117053)
REDDI A.hari Prof., Johns Hopkins University, School of Medicine, 医学部, 教授
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| Project Period (FY) |
1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1995: ¥3,800,000 (Direct Cost: ¥3,800,000)
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| Keywords | reparative dentin / pulpal wound healing / bone morphogenetic protein / dentin morphogenetic protein (DMP) / molecular cloning / polymerase chain reaction (PCR) |
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| Research Abstract |
Dental pulp cells have the potential to differentiate into odontoblasts during pulpal wound healing process. Demineralized dentin matrix induces reparative dentin formation in the amputated pulp and new bone in ectopic sites indicating the presence of bone morphogenetic protein (BMP) activity. We hypothesize that there is "dentin morphogenetic protein (DMP)" in dentin matrix belonging to BMPs and/or TGF-beta superfamily and they play a critical role in pulpal wound healing and reparative dentin formation. Therefore, we tried to identify and purify DMP.
Demineralized dentin matix was extracted in 4M quanidine HCl, loaded onto hydroxyapatite column and the 100 mM PO_4 eluate was loaded onto heparin-Sepharose CL-6B.The 0.5M NaCl eluate was then loaded in TSK-C18-4PW column by reversed phase high pressure liquid chromatography (HPLC). The collected samples were assayd by BMP activity using subcutaneous implantation. The active fraction was finally purified by Smart System reversed phase HPLC.The sample with a high alkaline phospatase stimulating activity was subjected to amino acied sequence analysis. The any sequences, however were not related to those of known TGF-beta superfamily members.
An alternative approach to the purification of novel DMP was the cloning of BMPs by polymerase chain reaction (PCR). The rat incisor pulp was extracted and poly A^+ RNA was prepared to synthesize first strand cDNA as templete. PCR was performed with SJL 151-159 and 187-201 as 3'primer, SJL 160 as 5'primer. We identified BMP-2, -4, -6, -7, -8, Inhibin-beta, Growth and Differentiation Factor (GDF) -1, -5, -6, -8 and -11 from 350 plasmid-clones using an automatic sequencer. We are now trying to localize these factors in embryonic tooth and reparative dentin by in situ hybridization to determine whether these factors were concerned in differentiation of odontoblasts.
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