| Co-Investigator(Kenkyū-buntansha) |
KANAKURA Yuzuru Osaka Univetsty, Graduate School of Medicine, Professor (20177489)
MIZUKI Masao Osaka Univetsty, Hospital, Associate Professor (80283761)
ORITANI Kenji Osaka Univetsty, Graduate School ofMedicine, Associate Professor (70324762)
SHIBAYAMA Hirohiko Osaka Univetsty, Graduate School of Medicine, Assistant Professor (60346202)
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| Research Abstract |
In the present study, we investigated the role of SIRT1 in hematopoietic stem cells.
At first, we examined the effects of SIRT1 inhibitor, nicotinamide(NA), and its activator, resveratol, on murine hematopoietic stem/progenitor cells. We isolated Lineage(-) Sca-1(+), c-kit(+) (KSL) cells from murine bone marrow and cultured with the cytokine cocktail containing SCF, IL-6, Flt3L, and TPO, which is utilized for the expansion of stem cells, together with NA or resveratol. As a result, NA significantly reduced LS cell population from 21.5% to 5.7%, while resveratol increased this fraction. Also, we performed colony assays using KSL cells cultured with or without NA for two days. The numbers of CFU-mix, BFU-E, CFU-E, CFU-G, CFU-M, and CFU-Meg yielded from NA-treated cells were all reduced about 50-80% as compared with those from untreated cells. We also examined the effects of NA on terminal differentiations of KSL cells. For this purpose, we cultured KSL cells using the following cytokine c
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ombinations : SCF and G-CSF for inducing granulocytic differentiation ; SCF and EPO for erythroid differentiation, or with SCF and TPO for megakaryocytic differentiation. NA accelerated differentiation toward all lineages. To inhibit SIRT1 activity more specifically, we introduced SiRNA for SIRT1 into murine KSL cells using the retrovirus system. In consistent with the results obtained from the experiments using NA, the proportion of immature KSL cells was reduced from 8.0% to 1.8%, and terminal differentiation was promoted in SIRT1 SiRNA-infected cells in comparison with MOCK-infected cells. We further examined the mechanisms through which SIRT1 keeps hematopoietic cells undifferentiated. We analized the gene expression profile in KSL cells under culture with or without NA. By RT-PCR, expressions of p16INK4A and pl9Arf were increased in NA-mediated cells. Furtheremore, we observed that ROS accumulation in NA-treated cells, and N-acetyl Cytosine(NAC) (an antioxidant) eliminated the differentiation promotion by NA. Thus, SIRT1 was supposed to regulate the fate and differentiation of hematopoietic cells through ROS reduction. Less
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