| Co-Investigator(Kenkyū-buntansha) |
TSUSHIMA Hideki Nagasaki University, Hospital of Medicine and Dentistry, Instructor, 医学部・歯学部附属病院, 助手 (70359959)
MIYAZAKI Yasushi Nagasaki University, Hospital of Medicine and Dentistry, Assistant Professor, 医学部・歯学部附属病院, 講師 (40304943)
TOMONAGA Masao Nagasaki University, Hospital of Medicine and Dentistry, Professor, 大学院・医歯薬学総合研究科, 教授 (40100854)
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| Budget Amount *help |
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2004: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
At first, we investigated frequency of side population (SP) cells in bone marrow (BM) mononuclear cells (MNC) of clinical cases with myelodysplastic syndrome (MDS : 24 cases), aplastic anemia (AA : 5 cases), and paroxysmal nocturnal hemoglobinuria (PNH : 3 cases). Mean values of SP cell population in MDS, AA, PNH, and healthy BM were 0.62%, 0.006%, 0.42%, and 0.23%, respectively. There was no significance in these frequencies between MDS, PNH, and healthy BM. With regard to WHO subtypes of MDS, we could detect the frequency of SP cells as follows ; RA 0-1.41%, RARS 0.03-0.57%, RCMD 0.11-2.04%, RAEB1 1.18-2.63%, and RAEB2 3.11% (one case). Next, to confirm clonality of SP cells in MDS samples, we sorted SP cells from two cases with trisomy 8 and one case of monosomy 7 by FACS cell sorter, and investigated their recurrent chromosomal abnormalities, which were observed in original samples, by fluorescence in situ hybridization (FISH). In all three cases, it was indicated that SP cell popu
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lation involved abnormal clones. Concurrently, expression of ABCG2 protein in BMMNC was tested. Mean values of ABCG2+ cells in BMMNC of MDS, AA, and PNH were 3.73%, 0.01%, and 1.99%, respectively. Results of ABCG2 expression analysis by FACS were more stable and indicated less difference in each sample and examination than those of SP cell assay. By multi-marker analysis by FACS, it was indicated that the population of SP cells and ABCG2+ cells of MDS and PNH expressed CD34 or CD133 simultaneously, but didn't expressed surface markers of more differentiated hematopopietic cells (CD19, CD3, and CD33). Therefore, to investigate properties of SP cells and ABCG2+ cells as hematopoietic stem cells (HSC), we applied long-term culture-initiated cells (LTC-IC) assay and cobblestone area forming cells (CAFC) assay to these populations. In AA cases, both LTC-IC and CAFC were markedly reduced. In MDS and PNH cases, the frequencies of LTC-IC and CAFC were lower than healthy BM. Furthermore, in MDS cases, cell numbers forming each CA were less than in healthy BM, and CAs in MDS samples were regressed earlier (in 3-5 weeks) than in healthy BM samples (5-7 weeks) by same stimulatory condition of growth factors. Taken together, quantitative and functional abnormalities of HSC in MDS/AA/PNH were indicated through the analysis of SP cell and ABCG+ cell populations. Less
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