| Co-Investigator(Kenkyū-buntansha) |
KIMURA Takafumi Department of Medicine, Kyoto Prefectural University of Medicine, Assistant, 医学部, 助手 (30275193)
OKUDA Tsukasa Department of Medicine, Kyoto Prefectural University of Medicine, Assistant Prof, 医学部, 講師 (30291587)
MURAKAMI Akira Department of Polymer Science, Kyoto Institute of Technology, Professor, 繊維学部, 教授 (60210001)
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| Budget Amount *help |
¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 1998: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1997: ¥3,700,000 (Direct Cost: ¥3,700,000)
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| Research Abstract |
Multicolor analysis of the expression of cell surface differentiation antigens, cytokine receptors, and gpl3O on BM, PBSC, and CB-derived CD34^+ cells revealed that the expression patterns differ between different stem/progenitor sources. First of all, we assessed the biologic role of signaling through gp13O, a signal-transducing receptor (R) component, in human hematopoiesis in vitro. Although PBSC-derived CD34^+ cells ubiquitously expressed gpl3O and IL-3Ralpha, IL-6Ralpha was only detected on 80% of these CD34^+ cells. We sorted CD34^+IL-6R^- cells and studied the effect on hematopoietic colony formation of signaling through gpl3O activated by a combination of IL-6 and recombinant soluble IL-6R (sIL-6R) in the presence of SCF and IL-3. Signals activated by SCF, IL-6/sIL-6R complex alone did not induce significant colony formation. However, a combination of IL-3, SCF, and IL-6/sIL-6R complex dramatically induced many neutrophil (CFU-G), erythroid-burst (BFU-E), mixed (CFU-Mix), and m
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egakaryocyte (CFU-Meg) colony formations. Our data suggested that LL-3 support was important forsurvival and initial proliferation, whereas SCF was important for enhancement of the proliferation of progenitors. The signal through gpl3O activated by IL-6/sIL-6R complex may mainly contribute to the maturation of progenitors in the presence of IL-3 and SCF.These results suggest that simultaneous activation of the three signals through gpl3O, c-kit, and IL-3R can induce in vitro proliferation and differentiation of trilineage hematopoietic progenitors in the absence of terminally acting lineage-specific factors.
Next, we tried to study molecular mechanisms of the crosstalks between these three signals. First, we established the method that allowed a great expansion of CFU-E in vitro. We obtained cell lysates from expanded CFU-Es, and analysed the tyrosine phosphorylation pattern after the stimulation of three signals or Epo. Simultaneously, we analysed the activation pattern of JAK-STAT pathway and MAPK pathway using immunoprecipitation and immunoblotting, and gel shift assay. Data accumulated suggest that activation of three signals may lead to cross-activation of additional signals. These molecular biologic analysis of signal transduction may provide a better understanding of interactions among signals through gpl3O and specific cytokine receptors and/or receptor type tyrosine kinases. Less
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