molecular cloning of self-renewal factor for hematopoietic stem cells and its clinical application

造血干细胞自我更新因子的分子克隆及其临床应用

• 批准号: 11357008
• 负责人: NAKAHATA Tatsutoshi
• 金额: $ 22.4万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11357008/
• 关键词: hematopoietic stem cells; self-renewal factor; AGM region; stromal cell line; molecular cloning; mRNA; mouse; novel genes; Ex vivo増幅; 臨床応用; 骨髄移植

Recent studies on the developing mouse embryo have shown that although primitive erythropoiesis is detectable in the yolk sac (YS) at 7.5 days post coitum (dpc), long-term repopulating hematopoietic stem cells (LTR-HSC) are first identified in the aorta-gonad-mesonephros (AGM) region at 10 dpc, prior to such activity being observed in YS and fetal liver, and expand in 11 dpc AGM region, suggesting that the AGM region at 10 to 11 dpc provides a microenvironment suitable for the development of LTR-HSC.These observations prompted us to establish stromal cell lines from the AGM region at 10 to 11 dpc, which would support ex vivo expansion of hematopoietic stem cells. We succeeded the establishment of a novel stromal cell line (AGMA9) from the AGM region of 10.5 dpc mouse embryo. When co-cultured with the AGMA9, lin-Sca-1+c-Kit+ cells isolated form adult mouse bone marrow and human cord blood CD34+ cells, significantly proliferated without additional cytokines. Expanded cocultured human CD34+ cells with AGMA9 for 4 weeks could reconstitute bone marrow of NOD/SCID mice suggesting that the cell line express some novel molecules which affect proliferation of not only murine but also human hematopoietic stem cells. This cell line can now be used to elucidate the molecular mechanisms regulating early hematopoiesis, and provide strategies for manipulation of primitive hematopoietic progenitor/stem cells. In RT-PCR analysis, AGMA9 produced detectable levels of SCF, SDF-1, OSM, IL-6, HGF, MIP-1 γ, Gas6, MCP-3, but no detectable levels of M-CSF EPO, TPO, Flk2/Flt3 ligand, MIP-1 α. We started molecular cloning of a novel self-renewal factor of hematopoietic stem cells using cDNA libraries of AGMA9 and AGMA7 which established from the AGM region at 10.5 dpc, and could not support proliferation of hematopoietic stem cells. We succeeded the cloning of more than ten novel genes and started the analysis of their functions.

关于发育中的小鼠胚胎的最新研究表明，尽管在涂层后7.5天（DPC）在卵黄囊（Ys）中可检测到原始的红细胞生成，但长期重植物的造血干细胞（LTR-HSC）在yorta-gonad-mesonephros（agm）区域中首次在10 dpc的earta-gonad-mesonephros区域中首次确定在11 DPC AGM区域中，表明10至11 dpc时的AGM区提供了适合LTR-HSC发展的微环境。这些观察结果促使我们在10到11 dpc以从AGM区域建立基质细胞系，这将支持造血干细胞的体内扩张。我们成功地建立了从10.5 dpc小鼠胚胎的AGM区域的新型基质细胞系（AGMA9）。当与AGMA9共培养时，LIN-SCA-1+ C-KIT+细胞分离出成年小鼠骨髓和人脐带血CD34+细胞，显着增殖而没有其他细胞因子。扩展的共培养的人CD34+与AGMA9延长了4周，可以重建点头/SCID小鼠的骨髓，这表明细胞系表达了一些新的分子，这些分子不仅影响鼠的增殖，而且还影响人类造血干细胞的增殖。现在，该细胞系可用于阐明调节早期造血的分子机制，并提供操纵原始造血祖细胞/干细胞的策略。在RT-PCR分析中，AGMA9产生可检测水平的SCF，SDF-1，OSM，IL-6，HGF，HGF，MIP-1γ，GAS6，MCP-3，但没有可检测到的M-CSF EPO，TPO，TPO，FLK2/FLK2/FLT3 IGAND3配体，MIP-1α。我们使用AGMA9和AGMA7的cDNA库开始了新型造血干细胞的新自我更新因子的分子克隆，该因子从AGM区域建立在10.5 dpc，无法支持造血干细胞的增殖。我们继承了十多种新型基因的克隆，并开始对其功能进行分析。

项目成果

Matsuoka S,Ebihara Y,Xu M,Ikeda Y,Nakahata T,Tsuji K: "CD34 expression on long-term repopulating hematopoietic stem cells changes during developmental stages"Blood. (in press).

Matsuoka S、Ebihara Y、Xu M、Ikeda Y、Nakahata T、Tsuji K：“长期再生造血干细胞在发育阶段的 CD34 表达变化”血液。

久保田優,濱畑啓悟,渡邉健一郎,林英蔚,小石誠二,宇佐美郁哉,中畑龍俊,秋山祐一: "小児赤白血病の3症例:予後因子の文献的考察"臨床血液. 41(3). 212-217 (2000)

Yu Kubota、Keigo Hamabata、Kenichiro Watanabe、Hidetaka Hayashi、Seiji Koishi、Ikuya Usami、Tatsutoshi Nakahata、Yuichi Akiyama：“儿童红白血病的三例：预后因素的文献综述”212-217（2000）。 ）

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 pre

Sugiyama H.、Nonaka T.、Kishimoto T.、Komoriya K.、Tsuji K.、Nakahata T.：“过氧化物酶体增殖物激活受体在人类培养的肥大细胞中表达：这些受体在肥大细胞负调节中的可能作用

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

Hasekawa S.、Pawankar R.、Suzuki K.、Nakahata T.、Furukawa S.、Okumura K.、Ra C.：“人血小板中 IgE 高亲和力受体 (Fc epsilon RI) 的功能表达及其细胞内表达

Yamashita T, Nakahayta T.: "Current knowledge on the pathophysiology of fanconi anemia : from genes to phenotypes"Int J Hematol. in press.

Yamashita T、Nakahayta T.：“范可尼贫血病理生理学的当前知识：从基因到表型”Int J Hematol。