Establishment of cardiac myocyte cell line

心肌细胞系的建立

• 批准号: 06557042
• 负责人: KOMURO Issei
• 金额: $ 10.56万
• 依托单位: 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: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557042/
• 关键词: embrvonic cell; transcription factor; cardiac development; MAP kinase; homeobox; zine finger; tumer suppresser gene; 心筋細胞; 心房性ナトリウム利尿ペプチド; 脳型クレアチンキナーゼ; 細胞移植; 細胞成長因子; 心筋細胞株

We have established cardiac differentiation system using embrryonic stem (ES) cells. When ES cells were cultured in suspension, they developed to embryoid bodies (EB) and started to beat spontaneously from 8 days. Northern blot analysis revealed that cardiac specific homeoprotein CSX and zinc finger protein GATA4 were expressed in EB from 5 days and contractile protein genes such as alpha myosin heavy chain and myosin light chain genes were expressed from 8 days after initiation of suspension culture. We examined the role of mitogen-activated protein kinase (MAPK) in cardiac differentiation using this system. Dominant negative MAPK strongly suppressed transcription of CSX and constitutively activated MAPK conversely activated transcription of CSX.We next isolated ES cell lines (designated as ES-MKP) which were permanently transfected by MAPK phosphatase (MKP), which is a negative regulator of MAPK.When ES-MKP was cultured in suspension, a few EB showed spontaneous contraction and expressed a little myosin heavy chain protein. These results suggest that MAPK is necessary for the development of cardiac myocytes.

我们建立了胚胎干细胞心肌分化体系。当ES细胞悬浮培养时，它们发育成胚状体（EB），并从第8天开始自发搏动。北方印迹分析显示，心脏特异性同源异型蛋白CSX和锌指蛋白GATA 4在EB中从5天开始表达，并且收缩蛋白基因如α肌球蛋白重链和肌球蛋白轻链基因从悬浮培养开始后8天开始表达。我们研究了丝裂原活化蛋白激酶（MAPK）在心脏分化中的作用。显性负性MAPK强烈抑制CSX的转录，而组成性激活的MAPK则相反激活CSX的转录，我们进一步分离了MAPK磷酸酶（MAPK phosphatase，MKP）永久转染的ES细胞系（ES-MKP），当ES-MKP悬浮培养时，少数EB出现自发收缩，并表达少量肌球蛋白重链蛋白。这些结果表明，MAPK是心肌细胞发育所必需的。

项目成果

小室一成,出雲正剛: "Cardiac-specific horeobox-Containirg Gene of the mause.in“Ceoelopment mechanisms of heart disease"" Markwald, RR., Clark, EB., Takao.A., 679 (1995)

Kazunari Komuro、Masatake Izumo：“心脏病的心脏特异性 horeobox-Containirg 基因。”Markwald, RR.、Clark, EB.、Takao.A., 679 (1995)

Komuro I.,Izumo S.: "Cardiac-specific homeobox-confaining gene of the mouse,in "Developmental mechanisms of heart disease"" Markwald R.R.,Clark E.B.,Takao A.Futura N.Y., 679 (1995)

Komuro I.，Izumo S.：“小鼠心脏特异性同源盒限制基因，在“心脏病的发展机制”中”Markwald R.R.，Clark E.B.，Takao A.Futura N.Y.，679（1995）

Yamazaki T.,Komuro I.et al.: "Endothelin-1 Is Involved in Mechanical Stress-induced Cardiomyocyte Hypertrophy" J Biol Chem. 271(6). 3221-3228 (1996)

Yamazaki T.，Komuro I.et al.：“Endothelin-1 参与机械应激诱导的心肌细胞肥大”J Biol Chem。

Komuro I,Izumo. S.: Cardiac-specific homeobox-containing gene of the mouse in Pevelopmental mechanisms of heart disease.Markwald R.R.Clack E.B.TakaoA Futura N.Y., (1995)

小室一世、出云。

Zou Y.,Komuro I.et al: "Protein Kinase C,but Not Tyrosine Kinases or Ras,Playsa Critical Role in Argiotensin U-induced Activation of Raf-1 Kinase and Extrocellular Signaly Protein Kinases in Cardiac Myocytes" J Biol Chem. 271(52). 33592-33597 (1996)

Zou Y.，Komuro I.等人：“蛋白激酶 C，但不是酪氨酸激酶或 Ras，在心肌细胞中精氨酸 U 诱导的 Raf-1 激酶和细胞外信号蛋白激酶激活中发挥关键作用”J Biol Chem。