2006 Fiscal Year Final Research Report Summary
Establishment of Vital Pulp Therapy using RNA interference Technique.
| Project/Area Number |
17591999
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Conservative dentistry
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| Research Institution | Ohu University |
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Principal Investigator |
YOKOSE Satoshi Ohu University, School of Dentistry, Professor, 歯学部, 教授 (90245803)
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| Project Period (FY) |
2005 – 2006
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| Keywords | RNAi / Dental pulp / Dentinogenesis / Lef-1 / Msx-1 |
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| Research Abstract |
Lef-1 is a member of HMG-domain protein, and thought to play important roles in inductive tissue interaction during tooth development. Loss of lef-1 mice demonstrated arrest at bud stage in tooth development. Furthermore, lef-1 participated in regulation of a large and diverse set of peptide growth factors in ectomesenchymal cells differentiation of dental papilla. These facts suggest that lef-1 can be a key factor influenced on odontoblast differentiation, but much about their precise information of relation between lef-1 and odontoblast differentiation has been unclear. To analyze the biological roles of lef-1 in regulating odontoblast differentiation, we examined the profiles of lef-1 expression during odontoblast differentiation of dental pulp cells in long-term mineralized cultures, and compared the expression pattern of lef-1 with those of dentin sialo protein (DSP), Osteocalcin, and Msx-1. Further, the expression vector inserted short interference RNA (siRNA) against either lef-
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1 or msx-1 was transfected in dental pulp cells. The efficiency of each siRNA sequences on target gene mRNA levels was determined by real time PCR. Dental pulp cells isolated from rat lower incisor were cultured for 25days. During culture period, the cells had differentiated into odontoblast-like cells, and demonstrated cellular peculiarity of odontoblast. Alkaline phosphatase activity was maximal on day 10, and decreased to day 25. The calcium contents in the cell layers were very low by day 10, and significantly increased from day 15. Dentin sialo protein (DST) mRNA expression increased for day 20, but remarkably decreased on day 25. Interestingly, the expression variations of DSP were analogous with those of lef-1 mRNA. Osteocalcin mRNA expression was very low by day 15, and significantly increased from day 20. Msx-1 mRNA expression was gradually increased and culminated by day 20. Both silence of lef-1 and msx-1 suppressed DSP expression and the mineralized formation. These results suggest that lef-1 regulate DSP expression in odontoblast-like cells and this mechanism is intimately correlated with msx-1 expression. The present study shows that lef-1 can be candidate for a key factor participating in odontoblast differentiation. Less
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