| Project/Area Number |
08670259
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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 |
Experimental pathology
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| Research Institution | Kyoto Prefectural University of Medicine (1997)
Sapporo Medical University (1996) |
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Principal Investigator |
OYAMADA Masahito Kyoto Prefectural University of Medicine, Department of Pathology, Assistant Professor, 医学部, 講師 (30183255)
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| Co-Investigator(Kenkyū-buntansha) |
OYAMADA Yumiko Kyoto Prefectural University of Medicine, Department of Pathology, Instructor, 医学部, 助手 (40231245)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1996: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Intercellular communication / Gap junctions / Connexin / Embyonic stem cells / Cardiogenesis / Gene targeting / 心筋細胞 / マイクロインジェクション / 細胞分化 / 心臓の発達 |
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| Research Abstract |
1.Connexin expression during cardiomyocytic differentiation of ES cells in vitro
The transcripts for C_x43 and C_x45 were detected in undifferentiated ES cells and in embryoid bodies before and after the appearance of beating cardiomyocytes. In contrast, C_x40 transcripts were not observed in undifferentiated ES cells, and were barely detectable in 3-and 5-day-old embryoid bodies. C_x40 transcripts significantly increased with the appearance of beating cells similar to those of cardiac-specific genes. Dye coupling was present among undifferentiated ES cells, pre-beating cells of embryoid body outgrowth and ES cell-derived beating cardiomyocytes. When dye was injected into a beating cell, dye spread was restricted to neighboring beating cells. Immunofluorescence demonstrated that C_x43 protein was localized not only in beating cells but also in surrounding non-beating cells, whereas myosin heavy chain alpha/beta was exclusively positive in the beating cells. These data suggest that the expression of multiple connexins is differentially regulated during the cardiomyocytic differentiation of ES cells in vitro and that C_x40 expression may be linked to early stages in cardiomyocytic differentiation.
2.Cardiomyocytic differentiation of C_x43-deficient ES cells in vitro
C_x43-deficient ES cells showed little dye coupling measured by Lucifer Yellow dye transfer assay, but there was cardiomyocytic differentiation in vitro, which was not significantly different from that of wild-type ES cells. RT-PCR revealed that the amounts of C_x40 and heart-specific gene transcripts significantly increased with the appearance of beating cells as with wild-type ES cells, whereas C_x43 transcripts were not detected in the C_x43-deficient cells. These data indicate that C_x43 is not essential for cardiomyocytic differentiation of ES cells in vitro and that expression of other connexins such as C_x40 and C_x45 is not influenced by the absence of C_x43 expression.
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