1999 Fiscal Year Annual Research Report

造血幹細胞自己複製因子の遺伝子クローニングとその臨床応用

Research Project

Project/Area Number	11357008
Research Category	Grant-in-Aid for Scientific Research (A)
Research Institution	Kyoto University
Principal Investigator	中畑龍俊京都大学, 大学院・医学研究科, 教授 (20110744)
Co-Investigator(Kenkyū-buntansha)	辻浩一郎東京大学, 医科学研究所, 助教授 (50179991) 依藤亨京都大学, 大学院・医学研究科, 助手 (60220779) 宇佐美郁哉京都大学, 大学院・医学研究科, 助手 (10301235)
Keywords	造血幹細胞 / 自己複製因子 / Ex vivo増幅 / 臨床応用 / AGM領域 / ストローマ細胞株 / 遺伝子クローニング / 骨髄移植
Research Abstract	近年、造血幹細胞のex vivo増幅が世界的に試みられているが未だ成功していない。その主たる要因は造血幹細胞の自己複製にかかわる分子が同定されていないためと考えられる。造血幹細胞自己複製因子について検討するため、胎生9日目のマウス卵黄嚢、胎生10.5日目のAGM領域、胎生12日目の胎児肝から数種類のストローマ細胞株を樹立した。マウス骨髄よりFACSを用いて分離した造血幹細胞の含まれる分画、lin^-Sca-1^+c-Kit^+細胞を、各ストローマ細胞との共培養前後で致死量放射線照射マウスに移植し、骨髄再構築能を見るという方法で骨髄造血幹細胞増幅支持能を持ったストローマ細胞を選別すると、そのうち造血幹細胞の発生部位であるAGM領域から樹立した細胞株(A9)が造血幹細胞の増殖を長期にわたり支持する活性を持っていた。各種ストローマ細胞株とヒト臍帯血より分離した造血幹細胞の含まれる分画、lin^-CD34^+CD38^-細胞をサイトカイン無添加で共培養前後でNOD/SCIDマウスヘ移植し、移植後3ケ月以上経った時点でマウス骨髄、末梢血中に存在するヒト細胞のを種々の単クローン抗体を用いて解析した。A9だけがヒト造血幹細胞の増殖を4週以上の長期にわたって支持できることが明らかとなった。以上の結果はA9は種を越えて造血幹細胞の増殖に関与する分子を発現していることを示している。そこで、造血幹細胞増幅支持能を持ったA9および支持能を持たないA7のそれぞれからmRNAを抽出し、一方向のcDNAライブラリーを作製した。種々の方法を用いて造血幹細胞自己複製因子のクローニングに着手している。現在、4種類の新規遺伝子が同定され、この遺伝子をA7に発現させ造血幹細胞増殖支持活性が獲得できるかどうか検討中である。

Research Products

(23 results)

All Other

All Publications (23 results)

[Publications] Sui X.,Tsuji K.,Ebihara Y.,Kishimoto T.,Nakahata T.: "Soluble IL-6 receptor with IL-6 stimulates megakaryopoiesis from human CD34+cells through gp130 signaling"Blood. 93. 2525-2532 (1999)
[Publications] Hasegawa S.,Pawankar R.,Suzuki K.,Nakahata T.,Furukawa S.,Okumura K.,Ra C.: "Functional expression of the high affinity receptor for IgE (Fc epsilon RI) in human Platelets and its intracellular expression in human megakaryocytes"Blood. 93. 2543-2551 (1999)
[Publications] Honda H.,Suzuki T.,Inaba T.,Tsuji K.,Nakahata T.,Yazaki Y.,Look T.,Hirai H.: "Expression of E2a-HLF chineric protein induced T-cell apotosis, B-cell maturation arrest, and development of acute lymphoblastic leukemia"Blood. 93. 2780-2790 (1999)
[Publications] Kampuraj D.,Saito H.,Kaneko A.,Fukagawa K.,Tsuji K.,Nakahata T.: "Characterization of mast-cell-committed progenitors present in human cord blood"Blood. 93. 3338-3346 (1999)
[Publications] Kobayashi M.,Ueda K.,Kojima S.,Shimbo T.,Nishihira H.,Nakahata T.: "Serum granulocyte colony-stimulating factor levels in patients with chronic neutropenia of childhood: Modulation of G-CSF levels by myeloid precursor cell mass"Brit. J. Haematol.. 105. 486-490 (1999)
[Publications] Taniguchi T.,Endo H.,Asano S.,Fujita T.,Nakahata T.,Motokura T.: "Expression of p21Cip1/Waf1/Sdi1 and p27 Kip1 cyclin-dependent kinase inhibitors during human hematopoiesis"Blood. 83. 4167-4178 (1999)
[Publications] Kinoshita T.,Sekiguchi T.,Xu M-J.,Ito Y.,Tsuji K.,Nakahata T.,Miyajima A.: "Hepatic differentiation induced by oncostatin M attenuates fetal liver hematopoiesis"Proc, Natl. Acad. Sci. USA. 96. 7265-7270 (1999)
[Publications] Yang FC.,Tsuji K.,Oda A.,Xu MJ.,Ebihara Y.,Watanabe S.,Ikeda Y.,Nakahata T.: "Effects of human granulocyle colony-stimulating factor (hG-CSF) on megakaryopoiesis and platelet function in hG-CSF receptor-transgenic mice"Blood. 94. 950-958 (1999)
[Publications] Matsumoto T.,Seki Y.,Kubo M.,Nakahata T.,Yamada S.,Yoshimura A.: "Suppression of STAT5 functions in liver,mammary grands, and Tcells in cytokine-inducible SH2 protein-1 (CISI) transgenic mice"Mol. Cell. Biol.. 19. 6396-6407 (1999)
[Publications] Ui M.,Takeda M.,Arai T.,Yamada K.,Nakahata T.,Tokino T.,Nakamura Y.,Iba H.: "Retrovirus vectors designed for efficient transduction of cytotoxic or cytostatic genes"Gene Therapy. 6. 1670-1678 (1999)
[Publications] Ishii T.,Nakahata T.,Maekawa T.: "Expression of SDF-1/PBSF receptor CXCR4 on CD34+ human bone marrow cellsis a phenotypic alteration for committed lymphoid progenitors"J. Immunol.. (in press).
[Publications] Kajiwara M.,Nonoyama S.,Eguchi M.,Imai K.,Ochs HD.,Nakahata T.,Yata J.: "In vitro megakaryopocyle differentiation from CD34 positive cells is defective in patients with Wiscott-Aldrich syndrome and X-linked thrombocytopenia"Brit.J.Haematol.. (in press).
[Publications] Takagi M.,Nakamura T.,Sawada T.,Nakahata T.,Yokota T.,Heike T.: "Chimeric GM-CSF-leukemia inhibitory factor (LIF) receptor can transduce expansion signals in primitive hematopoietic progenitors lacking IL-6Ra, IL-11Ra and gp130"Mol. Cell. Biol.. (in press).
[Publications] Ishiguro A.,Nakahata T.,Kato T.,Suzuki Y.,Shimbo T.: "Age-related changes in serum thrombopoietin in children: high levels in infants by one month of age"Pediatr, Res.. (in press).
[Publications] Futaki M.,Yamashita T.,Yabe M.,Kato S.,Asano S.,Nakahata T.: "The IVS4+4A-T mutation of the Fanconi Anemia gene FANCC is not associated with severe phenotype in Japanese patients"Blood. (in press).
[Publications] Matsuoka S.,Tsuji K.,Xu M.,Yang FC.,Kaneko K.,Kikuchi A.,Nakahata T.: "CD 34 expression on long-term repopulating hematopoietic stem cells changes during develomental stages"Blood. (in press).
[Publications] Ueda T.,Yoshino H.,Ebihara Y.,Ito M.,Tsuji K.,Nakahata T: "Ex vivo expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin interleukin-6 and soluble interleukin-6 receptor"J. Clin. Invest.. (in press).
[Publications] Hayashi Y.,Honma Y.,Niitsu N.,Taki T.,Tsuji K.,Nakahata T.: "SN-1, a novel leukemic cell line with t(11;16)(q23;p13): Myeloid characteristics and resistance to retinoids and vitamin D3"Cancer Res.. (in press).
[Publications] Sugiyama H.,Nonaka T.,Kishimoto T.,Komoriya K.,Tsuji K.,Nakahata T.: "Peroxisome proliferator-activated receptors negatively regurate mast cell activation"FEBS Letters. (in press).
[Publications] Sugiyama H.,Nonaka T.,Kishimoto T.,Komoriya K.,Tsuji K.,Nakahata T.: "Peroxisome proliferator-activated receptors are expressed in human cultured mast cells: a possible role for these receptors in negative regulation of mast cell activation"Blood. (in press).
[Publications] 中畑龍俊: "In vitroでの末梢血幹細胞の増幅.症例に学ぶG-CSFの臨床"医薬ジャーナル. 14 (1999)
[Publications] 中畑龍俊、二木真琴: "遺伝子治療開発研究ハンドブック"日本遺伝子治療学会編、エヌ・テイ・エス. 6 (1999)
[Publications] 辻浩一郎、中畑龍俊: "内科学教科書"文光堂(東京) (印刷中).

1999 Fiscal Year Annual Research Report

造血幹細胞自己複製因子の遺伝子クローニングとその臨床応用

Principal Investigator

中畑 龍俊 京都大学, 大学院・医学研究科, 教授 (20110744)

Research Products

[Publications] Sui X.,Tsuji K.,Ebihara Y.,Kishimoto T.,Nakahata T.: "Soluble IL-6 receptor with IL-6 stimulates megakaryopoiesis from human CD34+cells through gp130 signaling"Blood. 93. 2525-2532 (1999)

[Publications] Hasegawa S.,Pawankar R.,Suzuki K.,Nakahata T.,Furukawa S.,Okumura K.,Ra C.: "Functional expression of the high affinity receptor for IgE (Fc epsilon RI) in human Platelets and its intracellular expression in human megakaryocytes"Blood. 93. 2543-2551 (1999)

[Publications] Honda H.,Suzuki T.,Inaba T.,Tsuji K.,Nakahata T.,Yazaki Y.,Look T.,Hirai H.: "Expression of E2a-HLF chineric protein induced T-cell apotosis, B-cell maturation arrest, and development of acute lymphoblastic leukemia"Blood. 93. 2780-2790 (1999)

[Publications] Kampuraj D.,Saito H.,Kaneko A.,Fukagawa K.,Tsuji K.,Nakahata T.: "Characterization of mast-cell-committed progenitors present in human cord blood"Blood. 93. 3338-3346 (1999)

[Publications] Kobayashi M.,Ueda K.,Kojima S.,Shimbo T.,Nishihira H.,Nakahata T.: "Serum granulocyte colony-stimulating factor levels in patients with chronic neutropenia of childhood: Modulation of G-CSF levels by myeloid precursor cell mass"Brit. J. Haematol.. 105. 486-490 (1999)

[Publications] Taniguchi T.,Endo H.,Asano S.,Fujita T.,Nakahata T.,Motokura T.: "Expression of p21Cip1/Waf1/Sdi1 and p27 Kip1 cyclin-dependent kinase inhibitors during human hematopoiesis"Blood. 83. 4167-4178 (1999)

[Publications] Kinoshita T.,Sekiguchi T.,Xu M-J.,Ito Y.,Tsuji K.,Nakahata T.,Miyajima A.: "Hepatic differentiation induced by oncostatin M attenuates fetal liver hematopoiesis"Proc, Natl. Acad. Sci. USA. 96. 7265-7270 (1999)

[Publications] Yang FC.,Tsuji K.,Oda A.,Xu MJ.,Ebihara Y.,Watanabe S.,Ikeda Y.,Nakahata T.: "Effects of human granulocyle colony-stimulating factor (hG-CSF) on megakaryopoiesis and platelet function in hG-CSF receptor-transgenic mice"Blood. 94. 950-958 (1999)

[Publications] Matsumoto T.,Seki Y.,Kubo M.,Nakahata T.,Yamada S.,Yoshimura A.: "Suppression of STAT5 functions in liver,mammary grands, and Tcells in cytokine-inducible SH2 protein-1 (CISI) transgenic mice"Mol. Cell. Biol.. 19. 6396-6407 (1999)

[Publications] Ui M.,Takeda M.,Arai T.,Yamada K.,Nakahata T.,Tokino T.,Nakamura Y.,Iba H.: "Retrovirus vectors designed for efficient transduction of cytotoxic or cytostatic genes"Gene Therapy. 6. 1670-1678 (1999)

[Publications] Ishii T.,Nakahata T.,Maekawa T.: "Expression of SDF-1/PBSF receptor CXCR4 on CD34+ human bone marrow cellsis a phenotypic alteration for committed lymphoid progenitors"J. Immunol.. (in press).

[Publications] Kajiwara M.,Nonoyama S.,Eguchi M.,Imai K.,Ochs HD.,Nakahata T.,Yata J.: "In vitro megakaryopocyle differentiation from CD34 positive cells is defective in patients with Wiscott-Aldrich syndrome and X-linked thrombocytopenia"Brit.J.Haematol.. (in press).

[Publications] Takagi M.,Nakamura T.,Sawada T.,Nakahata T.,Yokota T.,Heike T.: "Chimeric GM-CSF-leukemia inhibitory factor (LIF) receptor can transduce expansion signals in primitive hematopoietic progenitors lacking IL-6Ra, IL-11Ra and gp130"Mol. Cell. Biol.. (in press).

[Publications] Ishiguro A.,Nakahata T.,Kato T.,Suzuki Y.,Shimbo T.: "Age-related changes in serum thrombopoietin in children: high levels in infants by one month of age"Pediatr, Res.. (in press).

[Publications] Futaki M.,Yamashita T.,Yabe M.,Kato S.,Asano S.,Nakahata T.: "The IVS4+4A-T mutation of the Fanconi Anemia gene FANCC is not associated with severe phenotype in Japanese patients"Blood. (in press).

[Publications] Matsuoka S.,Tsuji K.,Xu M.,Yang FC.,Kaneko K.,Kikuchi A.,Nakahata T.: "CD 34 expression on long-term repopulating hematopoietic stem cells changes during develomental stages"Blood. (in press).

[Publications] Ueda T.,Yoshino H.,Ebihara Y.,Ito M.,Tsuji K.,Nakahata T: "Ex vivo expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin interleukin-6 and soluble interleukin-6 receptor"J. Clin. Invest.. (in press).

[Publications] Hayashi Y.,Honma Y.,Niitsu N.,Taki T.,Tsuji K.,Nakahata T.: "SN-1, a novel leukemic cell line with t(11;16)(q23;p13): Myeloid characteristics and resistance to retinoids and vitamin D3"Cancer Res.. (in press).

[Publications] Sugiyama H.,Nonaka T.,Kishimoto T.,Komoriya K.,Tsuji K.,Nakahata T.: "Peroxisome proliferator-activated receptors negatively regurate mast cell activation"FEBS Letters. (in press).

[Publications] Sugiyama H.,Nonaka T.,Kishimoto T.,Komoriya K.,Tsuji K.,Nakahata T.: "Peroxisome proliferator-activated receptors are expressed in human cultured mast cells: a possible role for these receptors in negative regulation of mast cell activation"Blood. (in press).

[Publications] 中畑龍俊: "In vitroでの末梢血幹細胞の増幅.症例に学ぶG-CSFの臨床"医薬ジャーナル. 14 (1999)

[Publications] 中畑龍俊、二木真琴: "遺伝子治療開発研究ハンドブック"日本遺伝子治療学会編、エヌ・テイ・エス. 6 (1999)

[Publications] 辻浩一郎、中畑龍俊: "内科学教科書"文光堂(東京) (印刷中).

中畑龍俊京都大学, 大学院・医学研究科, 教授 (20110744)