Practical development of a novel method for hematopoietic stem cell expansion

造血干细胞扩增新方法的实际开发

• 批准号: 09357010
• 负责人: HIRAI Hisamaru
• 金额: $ 18.24万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09357010/
• 关键词: Notch; DSL domain; Jagged1; differentiation; active Notch1; transcription factor; GATA-2; Notch1; Notch2; Notch3; EGF様リピート; 造血幹細胞; 自己複製能; ストローマ細胞; ES細胞; 遺伝子治療; CD34; アデノウイルス; EGF受容体; LTC-IC

The Delta/Serrate/LAG-2 (DSL) domain-containing proteins are considered to be ligands for Notch receptors. However, the physical interaction between DSL proteins and Notch receptors is poorly understood. We found that mJagged1 physically bound to mouse Notch2 (mNotch2) on the cell surface and to a purified extracellular portion of mNotch2, respectively, in a CaィイD22+-ィエD2dependent manner. Deletion mutant analyses showed that the DSL Domain of mJagged1 is a minimal binding unit and is indispensable for binding to mNotch2. The solid-phase binding assay showed that Jagged1 binds to Notch1 and Notch3 in addition to Notch2, suggesting that mJagged1 is a ligand for multiple Notch receptors. We further analyzed the expression levels of transcription factors, C/EBP α, C/EBPε, PU.1, AML1b, c-myb, and GATA-2 as myeloid-specific transcription factors, and SCL, GATA-1, GATA-2, NF-E2 (p45), MafK (p18), EKLF, and c-myb as erythroid-specific transcription factors, before and after stimulation for differentiation of the wild-type and aNotch1-expressing 32D myeloid cells and F5-5 erythroid cells, respectively. As a result, the expression levels of those transcription factors except GATA-2 showed the same pattern in the wild-type and aNotch1-expressing cells, whereas the expression level of GATA-2 was found unchanged after stimulation for differentiation in the aNotch1-expressing 32D cells but decreased in the wild-type cells, suggesting inhibition of differentiation by aNotch1 is mediated through GATA-2.

含有Delta/Serrate/LAG-2（DSL）结构域的蛋白被认为是Notch受体的配体。然而，DSL蛋白和Notch受体之间的物理相互作用知之甚少。我们发现mJagged 1以Ca ~（2+）-NotchD 2依赖性方式分别与细胞表面的小鼠Notch 2（mNotch 2）和纯化的mNotch 2胞外部分物理结合。缺失突变体分析表明，DSL结构域mJagged 1是一个最小的结合单位，是必不可少的结合mNotch 2。固相结合试验表明，除了Notch 2，Jagged 1结合Notch 1和Notch 3，表明mJagged 1是多个Notch受体的配体。我们进一步分析了转录因子C/EBP α、C/EBPε、PU.1、AML 1b、c-myb和加塔-2作为骨髓特异性转录因子的表达水平，以及SCL、加塔-1、加塔-2、NF-E2（p45）、MafK（p18）、EKLF和c-myb作为红细胞特异性转录因子的表达水平。分别在刺激野生型和表达aNotch 1的32 D骨髓细胞和F5-5红细胞分化之前和之后。其结果是，在野生型和aNotch 1表达细胞中，除了加塔-2以外的转录因子的表达量显示出相同的模式，而在aNotch 1表达32 D细胞中，加塔-2的表达量在分化刺激后没有变化，但在野生型细胞中降低，表明aNotch 1对分化的抑制是通过加塔-2介导的。

项目成果

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