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The role of the TGF-B family in the cell differentiation from pluripotent bone marrow stem cells to immunocytes

TGF-B家族在多能骨髓干细胞向免疫细胞分化中的作用

基本信息

批准号：
17580262
负责人：
MURAKAMI Masaru
金额：
$ 2.32万
依托单位：
Azabu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17580262/
关键词：
TGF-β family signal transduction regulatory gene expression pluripotent stem cells mesodermal gene markers Mitf Brachyury(Bra)Goosecoid(Gsc)Nodal A6細胞 P19細胞 Brachyury Goosecoid nodal TGF-β activin goosecoid

项目摘要

The present study was examined to clarify the role of the TGF-β family in the cell differentiation from pluripotent bone marrow stem cells to immunocytes, essentially by using mouse P19 embryonal carcinoma cells which resembled the inner cell mass of the early embryo and had pluripotency, and A-6 which was a hematopoietic stem cell line established from mouse ES cells. When P19 cells were cultured in a tissue culture dish in the presence of serum, Brachyury (Bra) and Goosecoid (Gsc) were unexpectedly expressed. Expression of Bra and Gsc, mesodermal genes, was induced by the stimulation of the Nodal and Cripto pathway, indicating the importance of control of Nodal and Cripto expression for mesodermal formation. This Nodal signaling was also mediated via Smad2/3 through Alk4/5/7, mouse TGF-β type I receptors. Furthermore, it was suggested that unidentified molecules other than Lefty were involved in inhibition of the Nodal pathway during monolayer P19 cell culture, leading to down-regulation of Nodal and subsequently Bra and Gsc. In A-6 cells, there was significant gene expression of not only various TGF-β receptors, various Smads and undifferentiated markers but also tissue-specific transcription factors such as GATA-1, Mitf and so on. The examination of the expression and function of alternative splice variants of Mitf isoforms revealed a diverse pattern of gene expression and different transcriptional activities of Mitf isoforms, suggesting discrete regulation of gene transcription in specific tissues by Mitf. The expression and function of alternative splice variants of Alk5 were examined. Ubiquitous expression of Alk5 isoforms in mouse tissues and cells including A-6 cells and insignificant effects of alternative splicing on transcriptional activation and signaling induced by TGF-β were shown.

本研究主要通过使用小鼠P19胚胎癌细胞（其类似于早期胚胎的内细胞团并且具有多能性）和A-6（其是从小鼠ES细胞建立的造血干细胞系）来研究TGF-β家族在多能骨髓干细胞向免疫细胞的细胞分化中的作用。当P19细胞在血清存在下在组织培养皿中培养时，Brachyury（Bra）和Goosecoid（Gsc）出乎意料地表达。通过刺激Nodal和Cripto途径诱导中胚层基因Bra和Gsc的表达，表明控制Nodal和Cripto表达对中胚层形成的重要性。这种Nodal信号传导也通过Smad 2/3通过Alk 4/5/7（小鼠TGF-β I型受体）介导。此外，表明在单层P19细胞培养期间，除Lefty之外的未鉴定分子参与抑制Nodal途径，导致Nodal下调，随后是Bra和Gsc下调。在A-6细胞中，不仅有多种TGF-β受体、Smads和未分化标志物的基因表达，而且有组织特异性转录因子如加塔-1、Mitf等的基因表达。表明Mitf在特定组织中对基因转录的离散调节。检测Alk 5可变剪接变体的表达和功能。Alk 5同种型在小鼠组织和细胞（包括A-6细胞）中普遍表达，并且可变剪接对TGF-β诱导的转录激活和信号传导的影响不显著。