| Project/Area Number |
17590218
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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 pharmacology
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| Research Institution | Mie University |
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Principal Investigator |
OKAGAKI Tsuyoshi Mie University, Graduate School of Bioresouces, Assistant Professor, 大学院生物資源学研究科, 助教授 (80185412)
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| Co-Investigator(Kenkyū-buntansha) |
OOI Atsushi Mie University, Graduate School of Bioresouces, Research associate, 大学院生物資源学研究科, 助手 (70203693)
NAKAMURA Akio Gunma University, Graduate School of Medicine, lecturer, 大学院医学系研究科, 講師 (30282388)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2005: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | blood vessel / vascular smooth muscle cell / atherosclerosis / S100 family / 形質変換 / ミオシン / サブトラクション / 収縮型細胞 / 合成型細胞 |
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| Research Abstract |
Vascular smooth muscle cell (VSMC) shows two typical phenotype, contraction-type and synthetic-type upon various situations. We developed model system of phenotypic modulation by using cultured VSMC, P53LMAC01. Upon addition of PDGF, the VSMC changed to synthetic-type phenotype, and it changed to typical contraction-type phenotype in the presence of sodium butyrate (Na-B), which induce growth suppression by inhibiting acetylation of DNA. We isolated mRNA from these typical two types of cell and constructed subtraction cDNA library. We sequenced inserts of the library, and screened 230 clones from PDGF-specific library, and 160 clones from NaB-specific library. Further inserts of each clone were spotted to nylon membrane and examined by putative Northern hybridization to confirm the specificity in each types of cell. Four sets of membrane was hybridized with four sets of probes, two subtracted cDNAs and two original cDNAs from PDGF-and NaB-treated cells. As a result, 15 clones from PDGF-specific library and 19 clones from NaB-specific library were confirmed in the specificity of the expression. To quantify the expression level of these clones in PDGF-and NaB-treated cell, we made primer sets for each clone and examined with real time PCR. Clones showing 2-fold difference of expression among these two types of cell were S 100A4, S100A6, α-actin and others. We isolated the full-length cDNA of S 100A4 and S 100A6 by RT-PCR, and expressed these genes in bacteria. Using these gene products we affinity purified three proteins that interact with S 1004 and S100A6 proteins from the VSMC.
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