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Molecular cloning and characterization of differentiation-inducing factors into the cardiac myocyte lineage using an expression cloning approach

使用表达克隆方法对心肌细胞谱系的分化诱导因子进行分子克隆和表征

基本信息

批准号：
18590773
负责人：
UEYAMA Tomomi
金额：
$ 2.49万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590773/
关键词：
regenerative medicine stem cells cardiac myocyte differentiation-inducing factor cloning

项目摘要

Since embryonic stem (ES) cells are capable of self-renew and differentiation into specialized cells in response to appropriate signals, ES cells are proposed as a promising source of functional cardiac myocytes for cardiac diseases. However, to date the in vitro differentiation of ES cells into cardiac myocytes remains inefficient and its molecular mechanisms are not fully elucidated. Therefore, the identification of differentiation-inducing factors into the cardiac myocyte lineage has become critical for understanding the molecular mechanisms of differentiation into cardiac myocyte and heart development, and facilitates therapeutic applications of ES cells in cardiac diseases. In the present study, we sought to identify differentiation-inducing factors into the cardiac myocyte lineage using an expression cloning approach. We induced differentiation of the mouse embryonic carcinoma cell line, P19CL cells, into cardac myocytes using DMSO. We made cDNA libraries using mRNA isolated from differentiating cells. We then generated recombinant retroviruses expressing the cDNA libraries. ES cell clones that express EGFP under the transcriptional control of a cardiac-specific α-MHC promoter were infected with recombinant retroviruses expressing the cDNA libraries. Gemonic DNA was extracted from differentiated cells into cardiac myocytes and performed PCR to isolate the integrated cDNAs into the genomic DNA. We obtained G protein beta polypeptide 2 like 1 (Gnb2I1, Rack1), oxidase assembly 1-like (Oxa1l), glutaredoxin 5 homolog (Glrx5), and aurora kinase A interacting protein 1(Aurkaip1) as candidate genes for differentiation-inducing factors into the cardiac myocyte lineage. We are studying their functions in differentiation of ES cells into cardiac myocyte. Our study will provide novel insights into the molecular mechanisms of cardiac myocyte differentiation and the development of novel approaches for the directed differentiation of ES cells into cardiac myocytes.

由于胚胎干细胞（ES细胞）能够自我更新并响应适当的信号分化为特化细胞，因此ES细胞被认为是心脏疾病的功能性心肌细胞的有希望的来源。然而，迄今为止，ES细胞体外分化为心肌细胞的效率仍然很低，其分子机制尚未完全阐明。因此，鉴定心肌细胞谱系的分化诱导因子对于理解分化为心肌细胞和心脏发育的分子机制至关重要，并且有助于ES细胞在心脏疾病中的治疗应用。在本研究中，我们试图确定分化诱导因子的心肌细胞谱系使用表达克隆的方法。我们用DMSO诱导小鼠胚胎癌细胞系P19 CL细胞分化为心肌细胞。我们使用从分化细胞中分离的mRNA制备cDNA文库。然后我们产生了表达cDNA文库的重组逆转录病毒。用表达cDNA文库的重组逆转录病毒感染在心脏特异性α-MHC启动子的转录控制下表达EGFP的ES细胞克隆。从分化成心肌细胞的细胞中提取基因组DNA，并进行PCR以分离整合到基因组DNA中的cDNA。我们获得了G蛋白β多肽2样1（Gnb 2 I1，Rack 1），氧化酶组装1样（Oxa 11），谷氧还蛋白5同源物（Glrx 5）和极光激酶A相互作用蛋白1（Aurkaip 1）作为心肌细胞谱系分化诱导因子的候选基因。我们正在研究它们在ES细胞向心肌细胞分化中的作用。我们的研究将提供新的见解心肌细胞分化的分子机制和新的方法的发展，定向分化ES细胞向心肌细胞。

项目成果

期刊论文数量（0）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

MURC, a muscle-restricted coiled-coil protein that modulates the Rho/ROCK pathway, induces cardiac dysfunction and conduction disturbance

DOI：
10.1128/mcb.02186-07
发表时间：
2008-05-01
期刊：
MOLECULAR AND CELLULAR BIOLOGY
影响因子：
5.3
作者：
Ogata, Takehiro;Ueyama, Tomomi;Oh, Hidemasa
通讯作者：
Oh, Hidemasa

Single cardiac stem cells require stem cell antigen -1 to proliferate and survive for efficient cardiovascular regeneration

单个心脏干细胞需要干细胞抗原-1才能增殖和存活，以实现有效的心血管再生

DOI：
发表时间：
2007
期刊：
J Cell Sci 120
影响因子：
0
作者：
Tateishi;K.;Ashihara;E.;Takehara;N.;Nomura;T.;Honsho;S.;Nagagami;T.;Morikawa;S.;Takahashi;T.;Ueyama;T.;Matsubara;H.;Oh;H
通讯作者：
H

Overexpression of Toll-Like Receptors at the Vessel Wall Induces Atherosclerotic Lesion Formation. Synergism of TLR2 and TLR4

血管壁 Toll 样受体的过度表达诱导动脉粥样硬化病变形成。

DOI：
发表时间：
2007
期刊：
Arterioscler Thromh Vasc Biol 11
影响因子：
0
作者：
Shinohara;M.;Hirata;K.;Yamashita;T.;Takaya;T.;Sasaki;N.;Ueyama;T.;Emoto;N.;Inoue;N.;Yokoyama;M.;Kawashima;S
通讯作者：
S

Secreted phosphoprotein 1 enhances proliferative self-renewal through PI3K/Akt signaling in skeletal myosphere-derived progenitor cells.

分泌型磷蛋白 1 通过 PI3K/Akt 信号传导增强骨骼肌球来源的祖细胞的增殖自我更新。