ピルビン酸添加による骨髄単核球における血管内皮増殖因子の発現増強を目指した短期培養法の研究
血管内皮前駆細胞（EPC）を用いた血管再生療法は血管バイパス形成術や血管内膜切除術の適応とならない重症虚血肢に対する治療オプションとして近年脚光を浴びている．骨髄からのEPC動員や局所におけるEPCの血管内皮細胞への分化には血管内皮増殖因子（VEGF）が重要な役割を果たしていることから，今回我々はマウス骨髄単核球（BMMNC）を用いてVEGF遺伝子発現の増強およびVEGF分泌量の増加を図るための新規培養法の開発を目指し，以下の実験を行なった． VEGFは低酸素下で転写因子HIF-1が安定化することで誘導される．その背景にはプロリル水酸化酵素（HIF-PH）の活性がピルビン酸により阻害されること...
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| Published in | 日本輸血細胞治療学会誌 Vol. 58; no. 1; pp. 26 - 32 |
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| Main Authors | , , , , |
| Format | Journal Article |
| Language | Japanese |
| Published |
一般社団法人 日本輸血・細胞治療学会
2012
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| Subjects | |
| Online Access | Get full text |
| ISSN | 1881-3011 1883-0625 |
| DOI | 10.3925/jjtc.58.26 |
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| Abstract | 血管内皮前駆細胞（EPC）を用いた血管再生療法は血管バイパス形成術や血管内膜切除術の適応とならない重症虚血肢に対する治療オプションとして近年脚光を浴びている．骨髄からのEPC動員や局所におけるEPCの血管内皮細胞への分化には血管内皮増殖因子（VEGF）が重要な役割を果たしていることから，今回我々はマウス骨髄単核球（BMMNC）を用いてVEGF遺伝子発現の増強およびVEGF分泌量の増加を図るための新規培養法の開発を目指し，以下の実験を行なった． VEGFは低酸素下で転写因子HIF-1が安定化することで誘導される．その背景にはプロリル水酸化酵素（HIF-PH）の活性がピルビン酸により阻害されることが明らかになっている．ピルビン酸がBMMNCにおけるVEGF発現を増強することが期待されたため，マウスBMMNCのピルビン酸添加培養を行なった．EPC表面マーカーであるCD31+/CD34+二重陽性細胞は5mMピルビン酸により最も高くなり，VEGF遺伝子発現量は2日間の培養により培養前の27.8倍にまで達した．さらに培養液中VEGF濃度は5mMピルビン酸，4日間培養にて有意な上昇を認めた． ピルビン酸は細胞のエネルギー代謝上必須の有機酸であり，無害で安価のため，今回開発したEPC体外増幅法は今後治療的血管新生への応用が期待出来る． |
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| AbstractList | 血管内皮前駆細胞（EPC）を用いた血管再生療法は血管バイパス形成術や血管内膜切除術の適応とならない重症虚血肢に対する治療オプションとして近年脚光を浴びている．骨髄からのEPC動員や局所におけるEPCの血管内皮細胞への分化には血管内皮増殖因子（VEGF）が重要な役割を果たしていることから，今回我々はマウス骨髄単核球（BMMNC）を用いてVEGF遺伝子発現の増強およびVEGF分泌量の増加を図るための新規培養法の開発を目指し，以下の実験を行なった． VEGFは低酸素下で転写因子HIF-1が安定化することで誘導される．その背景にはプロリル水酸化酵素（HIF-PH）の活性がピルビン酸により阻害されることが明らかになっている．ピルビン酸がBMMNCにおけるVEGF発現を増強することが期待されたため，マウスBMMNCのピルビン酸添加培養を行なった．EPC表面マーカーであるCD31+/CD34+二重陽性細胞は5mMピルビン酸により最も高くなり，VEGF遺伝子発現量は2日間の培養により培養前の27.8倍にまで達した．さらに培養液中VEGF濃度は5mMピルビン酸，4日間培養にて有意な上昇を認めた． ピルビン酸は細胞のエネルギー代謝上必須の有機酸であり，無害で安価のため，今回開発したEPC体外増幅法は今後治療的血管新生への応用が期待出来る． |
| Author | 小倉, 浩美 菅野, 仁 入部, 雄司 藤井, 寿一 青木, 貴子 |
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| References_xml | – reference: 21) Dennis JE, Esterly K, Awadallah A, et al: Clinical-scale expansion of a mixed population of bone-marrow-derived stem and progenitor cells for potential use in bone-tissue regeneration. Stem Cells, 25: 2575-2582, 2007. – reference: 7) Kawamoto A, Katayama M, Handa N, et al: Intramuscular transplantation of G-CSF-mobilized CD34(+) cells in patients with critical limb ischemia: a phase I/IIa, multicenter, single-blinded, dose-escalation clinical trial. Stem Cells, 27: 2857-2864, 2009. – reference: 17) Plouffe BD, Kniazeva T, Mayer JE, et al: Development of microfluidics as endothelial progenitor cell capture technology for cardiovascular tissue engineering and diagnostic medicine. FASEB J, 23: 3309-3314, 2009. – reference: 18) Lee MS, Moon EJ, Lee SW, et al: Angiogenic activity of pyruvic acid in in vivo and in vitro angiogenesis models. Cancer Res, 61: 3290-3293, 2001. – reference: 4) Rehman J, Li J, Orschell CM, et al: Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation, 107: 1164-1169, 2003. – reference: 11) Akita T, Murohara T, Ikeda H, et al: Hypoxic preconditioning augments efficacy of human endothelial progenitor cells for therapeutic neovascularization. Lab Invest, 83: 65-73, 2003. – reference: 20) Powell RJ, Comerota AJ, Berceli SA, et al: Interim analysis results from the RESTORE-CLI, a randomized, double-blind multicenter phase II trial comparing expanded autologous bone marrow-derived tissue repair cells and placebo in patients with critical limb ischemia. J Vasc Surg, 54: 1032-1041, 2011. – reference: 12) Ivan M, Haberberger T, Gervasi DC, et al: Biochemical purification and pharmacological inhibition of a mammalian prolyl hydroxylase acting on hypoxia-inducible factor. Proc Natl Acad Sci U S A, 99: 13459-13464, 2002. – reference: 2) Kinnaird T, Stabile E, Burnett MS, et al: Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation, 109: 1543-1549, 2004. – reference: 8) Levine AJ, Puzio-Kuter AM: The control of the metabolic switch in cancers by oncogenes and tumor suppressor genes. Science, 330: 1340-1344, 2010. – reference: 1) Lawall H, Bramlage P, Amann B: Stem cell and progenitor cell therapy in peripheral artery disease. A critical appraisal. Thromb Haemost, 103: 696-709, 2010. – reference: 5) Tateno K, Minamino T, Toko H, et al: Critical roles of muscle-secreted angiogenic factors in therapeutic neovascularization. Circ Res, 98: 1194-1202, 2006. – reference: 6) Matoba S, Tatsumi T, Murohara T, et al: Long-term clinical outcome after intramuscular implantation of bone marrow mononuclear cells (Therapeutic Angiogenesis by Cell Transplantation [TACT] trial) in patients with chronic limb ischemia. Am Heart J, 156: 1010-1018, 2008. – reference: 19) Di Santo S, Yang Z, Wyler von Ballmoos M, et al: Novel cell-free strategy for therapeutic angiogenesis: in vitro generated conditioned medium can replace progenitor cell transplantation. PLoS One, 4: e5643, 2009. – reference: 14) Oyama H, Minakami S: Studies on erythrocyte glycolysis. V. Change of the glycolytic intermediate pattern of reticulocytes during maturation. J Biochem, 61: 103-107, 1967. – reference: 16) Krenning G, van Luyn MJ, Harmsen MC: Endothelial progenitor cell-based neovascularization: implications for therapy. Trends Mol Med, 15: 180-189, 2009. – reference: 3) Ziegelhoeffer T, Fernandez B, Kostin S, et al: Bone marrow-derived cells do not incorporate into the adult growing vasculature. Circ Res, 94: 230-238, 2004. – reference: 13) Aisaki K, Kanno H, Oyaizu N, et al: Apoptotic changes precede mitochondrial dysfunction in red cell-type pyruvate kinase mutant mouse erythroleukemia cell lines. Jpn J Cancer Res, 91: 171-179, 1999. – reference: 9) Semenza GL: Hydroxylation of HIF-1: oxygen sensing at the molecular level. Physiology (Bethesda), 19: 176-182, 2004. – reference: 10) Lu H, Dalgard CL, Mohyeldin A, et al: Reversible inactivation of HIF-1 prolyl hydroxylases allows cell metabolism to control basal HIF-1. J Biol Chem, 280: 41928-41939, 2005. – reference: 15) Aicher A, Zeiher AM, Dimmeler S: Mobilizing endothelial progenitor cells. Hypertension, 45: 321-325, 2005. |
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| SubjectTerms | 再生医療 血管内皮前駆細胞 血管内皮細胞増殖因子（VEGF） 重症虚血肢 |
| Title | ピルビン酸添加による骨髄単核球における血管内皮増殖因子の発現増強を目指した短期培養法の研究 |
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| ispartofPNX | 日本輸血細胞治療学会誌, 2012, Vol.58(1), pp.26-32 |
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