| Project/Area Number |
09671231
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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 |
General surgery
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| Research Institution | Yamaguchi University |
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Principal Investigator |
ZEMPO Nobuya Yamaguchi Univ.Sch.of Medicine, Assoc.Prof., 医学部, 助教授 (00206666)
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| Co-Investigator(Kenkyū-buntansha) |
FUJIOKA Kentaro Yamaguchi University Hospital, Instructor, 医学部附属病院, 助手 (80238542)
ESATO Kensuke Yamaguchi Univ.Sch.of Medicine, Professor, 医学部, 教授 (10034943)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1998: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1997: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | intimal hyperplasia / MT-MMP-1 / RT-PCR / antisense oligonucleotide / gene therapy |
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| Research Abstract |
We explored membrane-type matrix metalloproteinase- 1 (MT-MMP- 1) gene expressions in balloon-injured rat carotid artery, and that whether antisense MT-MMP-1 oligonucleotide inhibits cultured smooth muscle cell (SMC) proliferation and migration. The MT-MMP-1 mRNA expressions were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The primers for the amplification of MT-MMP-1 was designed as follows ; sense (5'-CCC TAT GCC TAC ATC CGT GA-3') and antisense (5'-TCC ATC CAT CAC TTG GTT AT-3'). SMC proliferation was measured by MTT assay, and the migration (chemoinvasion ) was assayed by Boyden chamber method. The optimal number of PCR cycles for MT-MMP-1 arid GAPDH was determined as 28 and 22, respectively. The MT-MMP-1 mRNA expression was faint in uninjured carotid arteries (control). The gene expression was increased on day 1 and showed to be maximal on day 4. It was gradually decreased thereafter. The relative radioactivity calculated by dividing the radioactivity associated with MT-MMP-l PCR products by that associated with the GAPDH gene products was 23 in control, 80 on day 1, 164 on day 4, 136 on day 8, and 54 on day 14. The antisense MT-MMIP-l oligonucleotides were designed for the SMC proliferation and migration assays as follows ; antisense 1 (5'-AGA CAG GGT CCC CGG CGT CG-3') and antisense 2 (5'-AGA CAG CAA GGA CCA CTG CC-3'). By using the antisense oligonucleotides, we are now conducting the SMC proliferation and migration assays. These results suggest that MT-MMP- 1 mRNA expression was increased at maximum 4 days after balloon injury when SMCs start to migrate from the media to the intima. Gene therapy with the antisense MT-MMP-1 oligonucleotides might inhibit SMC proliferation and migration in vitro and in vivo.
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