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Cell transplantation to myocadial infarction area

心肌梗死区细胞移植

基本信息

批准号：
12470267
负责人：
SAI Sadahiro
金额：
$ 6.91万
依托单位：
Tohoku 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

项目摘要

The purpose of the present study was to evaluate the application of cell transplantation into myocardium in a rodent infarction model. Embryonic stem (ES) cells were cultured and induced to differentiate inadvertently, and cells expressing myocardial phenotype revealed. We postulated that ES cells differentiating to cardiomyocytes expressed alpha-myosin heavy chain. We constructed an expression gene that consisted of alpha-myosin heavy chain promoter and cDNA encoding EGFP-IRES-zeocin and puromycin resistant gene, and this gene was transfected into ES cells. ES cells transfected with the gene were cultured in medium supplemented with zeoncin, and the transfected clones were determined by PCR. Cells were cultured in medium not supplemented with human leukemia inhibitory factor and differentiated to form embryoidbody. Some cells of the embryoidbody started beating spontaneously. EGFP expressing cells were existed but we could not detect enough number of the cells transfected with the gen … More e by flowcytometric analysis. We performed puromycin selection alternatively, but the selection was not effective to detect enough number of beating cells. Since the specific master genes for the differentiation to myocardial cells were not detected. To isolate the myocardial-phenotype cells from cultured deferentiated ES cells seemed to be a promising strategy for us. However, the transfected gene did not activated well in ES cells unexpectedly. The transfected gene seemed to be tuned off by unknown gene inactivation mechanism. It may be a methylation of transduced promoter gene to be speculated as one of the possible mechanism of the inactivation. To evaluate the effectiveness of the cell transplantation, we used cryo-injured model. Myocardium of Fisher rat were injured with a cryogenic probe and regional movement of the left ventricle were analyzed by using ultrasonic crystal probes. The accuracy of the measurement technique, however, was limited for the detection of reduction in the cardiac function. Less

本研究旨在探讨细胞移植在啮齿类动物心肌梗死模型中的应用。胚胎干细胞（ES）在不经意间被培养和诱导分化，细胞表达心肌表型。我们假设胚胎干细胞向心肌细胞分化时表达α -肌球蛋白重链。我们构建了由α -肌球蛋白重链启动子和编码EGFP-IRES-zeocin和puromycin耐药基因的cDNA组成的表达基因，并将该基因转染到ES细胞中。将转染该基因的胚胎干细胞置于添加zeoncin的培养基中培养，用PCR法测定转染的克隆。细胞在不添加人白血病抑制因子的培养基中培养，分化成胚状体。胚状体的一些细胞开始自发地跳动。存在表达EGFP的细胞，但流式细胞术检测到转染了EGFP的细胞数量不足。我们进行了嘌呤霉素的选择性选择，但这种选择不能有效地检测到足够数量的跳动细胞。由于没有检测到向心肌细胞分化的特异性主基因。从体外培养的胚胎干细胞分化中分离心肌表型细胞似乎是一种很有前途的策略。然而，转染的基因在胚胎干细胞中意外地没有很好地激活。转染的基因似乎由于未知的基因失活机制而被关闭。它可能是一个甲基化的转导启动子基因被推测为失活的可能机制之一。为了评估细胞移植的有效性，我们采用了冷冻损伤模型。用低温探针损伤Fisher大鼠心肌，用超声晶体探针分析左心室局部运动。然而，测量技术的准确性在检测心功能降低方面受到限制。少