权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Manipulation of hematopoietic cells for regenerative medicine

再生医学中造血细胞的操作

基本信息

批准号：
12557080
负责人：
NAKANO Toru
金额：
$ 8.58万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12557080/
关键词：
Hematopoiesis Embryonic Stem Cell Cell Differentiation Transcription Factor GATA-2 Gene Regulation Stroma Cell 細胞分化 GATA転写因子 Myb転写因子

项目摘要

We had established an in vitro differentiation induction method from mouse embryonic stem (ES) cells to hematopoietic cells by co-culturing the ES cells on OP9 stroma cell line (OP9 system). Although OP9 system can give rise to multipotential hematopoietic progenitors, hematopoietic stem cells with self-renewing activity never emerge. To improve the hematopoietic activity, we introduced conditional gene expression method to OP9 system. Combination of tetracycline gene regulating system (Tet-Off system) and OP9 system enabled us to express the desirable genes in the hematopoietic progenitor cells which are developed by OP9 system.Function of a zinc-finger type transcription factor GATA-2 was examined by the system. When GATA-2 was over-expressed, the numbers of hematopoietic colonies and the percentages of immature hematopoietic cells in individual colonies increased to 10 fold. Previous reports had suggested that GATA-2 inhibited the terminal differentiation of blood cells. However, our data was opposite ; i.e., terminal differentiation into erythroid and megakaryocyte lineages were enhanced. We speculated that this discrepancy should have been due to the adopted experimental system, since the previous studies used the fusion protein of GATA-2 and the ligand binding domain of estrogen receptor (GATA-2/ER) for conditional activation of the transcription factor instead of the authentic GATA-2. Then, we compared the biological functions of GATA-2 and GATA-2/ER. Surprisingly, the function of GATA-2 was opposite to that of GATA-2/ER. Binding capacity and inhibitory transcriptional interaction of GATA-2 to the other hematopoietic transcription factors, PU.1 and c-Myb, were quite different from those of GATA-2/ER.This study reveals the utility of the combination of OP9 system and Tet-Off system to investigate the molecular mechanisms of hematopoietic differentiation and for the future regenerative medicine.

本研究建立了小鼠胚胎干细胞（ES细胞）在OP 9基质细胞系（OP 9系统）上体外诱导分化为造血细胞的方法。尽管OP 9系统可以产生多能造血祖细胞，但具有自我更新活性的造血干细胞却从未出现。为了提高造血活性，我们在OP 9系统中引入了条件基因表达方法。将四环素基因调控系统（Tet-Off系统）与OP 9系统相结合，使目的基因在OP 9系统培养的造血祖细胞中表达，并对锌指型转录因子加塔-2的功能进行了研究。当过表达加塔-2时，造血集落的数量和单个集落中未成熟造血细胞的百分比增加至10倍。以前的研究表明，加塔-2抑制血细胞的终末分化。然而，我们的数据是相反的;即，向红系和巨核系的终末分化增强。我们推测，这种差异应该是由于所采用的实验系统，因为以前的研究使用的融合蛋白的加塔-2和雌激素受体的配体结合结构域（加塔-2/ER）的转录因子的条件激活，而不是真正的加塔-2。比较了加塔-2和加塔-2/ER的生物学功能。令人惊讶的是，加塔-2的功能与加塔-2/ER相反。加塔-2与其他造血转录因子PU.1和c-Myb的结合能力和抑制转录相互作用与加塔-2/ER有很大不同。