2000 Fiscal Year Final Research Report Summary
Cloning and Analysis of the Gene Induced by Cyclic Stretch Stimuli
| Project/Area Number |
11671418
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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 |
Orthopaedic surgery
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| Research Institution | Chiba University |
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Principal Investigator |
WADA Yuichi Chiba University, University Hospital, Assistant, 医学部・附属病院, 助手 (10282485)
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| Co-Investigator(Kenkyū-buntansha) |
SASHO Takahisa Chiba University, University Hospital, Assistant, 医学部・附属病院, 助手 (20312952)
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| Project Period (FY) |
1999 – 2000
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| Keywords | Mechnical stimuli / Mechano-receptor / Differential display / Cell membrane / Gene cloning |
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| Research Abstract |
A gene specifically induced by cyclic mechanical stimuli was cloned using differential display. Osteoblastic cell-line MC3T3E.1 was cultured under cyclic mechanical stimuli or statically. RNA of two different sources were extracted and used as the templates for differential display. Only one fragment was significantly highly expressed in cells cultured with mechanical stimuli in northern blot out of 5 fragments. The length of gene fragment was 206 base pairs. This fragment was used for the probe for further analysis. Full length of this gene found out to be 7.0bp. We determined the DNA sequence of this gene up to 2.5 kbp from 3' terminus. Homology search revealed this gene was a novel gene and a member of cell membrane bound receptor that penetrates cell membrane 7 times. RNA blot revealed this gene was expressed in the heart, lung, skeletel muscles where cells were exposed to cyclic mechanical stimuli. We named this gene KEYAKI (Kinetics evoked and kinetics induced) gene.
We examined in vivo pattern of expression using torn Achilles tendon rabbit. Intact tendon, fresh torn tendon and 4 weeks after tendon cut correspond to physiological level of stimuli, no stimuli, and recovered some level of stimuli respectively. The amount of expression was examined by semiquantitative RT-PCR.As a result, expression level of KEYAKI increased after the Achilles tendon was cut and decreased after the completion of healing of tendon.
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