| Project/Area Number |
16590688
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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 |
Circulatory organs internal medicine
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| Research Institution | Yamaguchi University |
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Principal Investigator |
MIURA Toshiro Yamaguchi University, Faculty of Medicine, Associate Professor, 医学部, 講師 (00243634)
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| Co-Investigator(Kenkyū-buntansha) |
IKEDA Yasuhiro Yamaguchi University, Faculty of Medicine, Assistant Professor, 医学部, 助手 (00260349)
HAMANO Koichi Yamaguchi University, Faculty of Medicine, Professor, 医学部, 教授 (60263787)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | tissue stem cell / cardiomyocyte / calcein / tongue muscle / NKX2.5 / Connexin 43 / intracellular calcium / gap junction / NMX2.5 / 幹細胞 / 心臓 / 圧負荷 / 分化 |
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| Research Abstract |
Tissue stem cells from mice (C57BL6,6-8W) heart, skeletal muscle, and tongue muscle were isolated by the magnetic cell sorting system with the reference of cell surface stem cell antigen-1(SCA-1). Tissues were dissolved in 0.2% collagenase solution and SCA-1 biotinylated monoclonal antibody were mixed and then anti-biotin monoclonal antibody with magnetic beads were reacted. The cells were passed through the magnetic column and the SCA-1(+) cells were isolated. The purity of the isolated cell of this method was more than 90% by comparing the fluorescent activated cell sorting (FACS). Cells were cultured in DMEM with 10% FCS with supplementation of EGF,PDGFbb, and LIF for 2 weeks. At 10 days, the cells from tongue muscle spontaneously beat and the calcium transient can be recorded. In other cells, there were scarce cells beating spontaneously. At 2 weeks, NKX2.5, Cx 43, ANP were expressed in the cells from tongue muscle by RT-PCR. Cx43 expression was also observed by Western blotting and immunohistochemistry. The functional gap junction formation was observed by the Calcein dye transfer method. We also observed the effect of pressure overload subjected to the high air pressure chamber for a week. The cardiac specific transcription factor NKX2.5 were more rapidly expressed in the cells with pressure overload. So the pressure overload is an physiological external stimuli to promote tissue stem cell differentiation to the cardiomyocyte lineage.
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