Development of regenerated cardiomyocyte for cardiovascular tissue engineering

心血管组织工程再生心肌细胞的开发

• 批准号: 11838016
• 负责人: FUKUDA Keiichi
• 金额: $ 2.69万
• 依托单位: KEIO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11838016/
• 关键词: bone marrow; stem cell; differentiation; cardiomyocyte; transplantatioN; 間葉系未分化幹細胞; 骨髄; 細胞移植; 心筋細胞; 心不全

We have isolated a cardiomyogenic cell line (CMG cell) from murine bone marrow stromal cells. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneous beating cells were repeatedly screened for. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine treatment in approximately 30% of the cells ; they connected with adjoining cells after 1 week, began spontaneous beating after 2 weeks, and beat synchronously after 3 weeks. They expressed ANP and BNP, and were stained with anti-myosin, -desmin and -actinin antibodies. Electron microscopy revealed a cardiomyocyte-like ultrastructure including typical sarcomeres, a centrally positioned nucleus, and atrial granules. These cells had several types of action potentials ; sinus node-like and ventricular cell-like action potentials. All cells had a long action potential duration or plateau, a relatively shallow resting membrane potential, and a pacemaker-like late diastolic slow depolarization. Analysis of the isoform of contractile protein genes, such as myosin heavy chain, myosin light chain and α-actin, indicated that their muscle phenotype was similar to fetal ventricular cardiomyocytes. These cells expressed Nkx2.5/Csx, GATA4, TEF-1 and MEF-2C mRNA before 5-azacytidine treatment, and expressed MEF-2A and MEF-2D after treatment. They expressed adrenergic α1A, α1B and α1D receptor before 5-azacytidine treatment, and expressed adrenergic β1, β2 and muscarinic M1 and M2 receptors after the treatment. Stimulation with phenylephrine, isproterenol and carbachol increased ERK phosphorylation and second messengers. Isoproterenol increased the beating rate, which was blocked with propranolol and CGP20712A (β_1-selective blocker) by 79% and 71%, respectively. This new cell line provides a powerful model for the study of cardiomyocyte differentiation and cardiovascular tissue engineering.

我们从鼠骨髓基质细胞中具有杂型细胞系（CMG细胞）。大约30％的细胞，并在3周后击败同步。颗粒。肌动蛋白表明，肌肉表型与触发性心肌细胞相似。 5-氮杂丁胺处理前的αD受体，并在随后的肾上腺素，Isproterenol和carbachol后表达肾上腺素能的β1，β2和毒蕈碱M1 EPTOR，ERK磷酸化和第二个eNOL增加了eNOL，这增加了blove速率。用于研究心肌细胞分化和心血管组织工程的79％和71％EL。

项目成果

K.Fukada,S.Makino,S.Ogawa. Et al.: "A Cardiomyocyte Cell Line To Overcome Fibrotic Myocardium."Microcirculation annual. 16. 17-18 (2000)

K.深田，S.牧野，S.小川。

Keiichi Fukuda, Shinji Makino: "Establishment of Cardiomyogenic Cell Line from Marrow Stroma"(Y,Shimizu Edit.) Elsevier. (2000)

Keiichi Fukuda、Shinji Makino：“从骨髓基质建立心肌细胞系”（Y，清水编辑）Elsevier。

Shinji Makino, Keiichi Fukuda: "Cardiomyocytes can be generated from marrow stromal cells in vitro"Journal of Clinical Investigation. 103. 697-705 (1999)

Shinji Makino、Keiichi Fukuda：“心肌细胞可以在体外由骨髓基质细胞产生”临床研究杂志。

M.Sano,K.Fukuda,H.Kodama,S.Tahara,S.Ogawa. Et al: "Autocrine/Paracrine Secretion of IL-6 Family Cytokines Causes Angiotensin II-Induced Delayed STAT3 Activation."Biochem.Biphys.Res.Com.. 269. 798-802 (2000)

M.Sano，K.Fukuda，H.Kodama，S.Tahara，S.Okawa。

H.Kodama,K.Fukuda,J.Pan,M.Murata,S.Ogawa. Et al.: "Significance of Raf-1/MEK/ERK cascade compared with JAK/STAT and PI3-K pathway in gp130-mediated Cardiac Hypertrophy."AM.J.Physiol.. 279. H1635-H1644 (2000)

H.Kodama，K.Fukuda，J.Pan，M.Murata，S.Okawa。