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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

基本信息

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

项目摘要

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尚不清楚。我们发现mJagged1分别在细胞表面与小鼠Notch2 （mNotch2）和纯化的mNotch2细胞外部分以Ca D22+- d2依赖的方式物理结合。缺失突变体分析表明，mJagged1的DSL结构域是最小的结合单元，是与mNotch2结合所必需的。固相结合实验表明，除了Notch2外，Jagged1还与Notch1和Notch3结合，表明mJagged1是多种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、AML1b、C -myb和GATA-2作为骨髓特异性转录因子，以及SCL、GATA-1、GATA-2、NF-E2 （p45）、MafK （p18）、EKLF和C -myb作为红细胞特异性转录因子，分别在刺激分化野生型和anotch1表达的32D髓细胞和F5-5红细胞前后的表达水平。结果表明，除GATA-2外，其他转录因子在野生型和aNotch1表达细胞中的表达规律相同，而在aNotch1表达的32D细胞中，刺激分化后GATA-2的表达水平没有变化，而在野生型细胞中表达水平下降，说明aNotch1对分化的抑制是通过GATA-2介导的。