Cell transplantation to myocadial infarction area

本研究的目的是评价细胞移植在大鼠心肌梗死模型中的应用。不经意间培养诱导胚胎干细胞分化，发现表达心肌表型的细胞。我们推测分化为心肌细胞的ES细胞表达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和扑霉素抗性基因的cDNA组成的表达基因，并将该基因导入ES细胞。将该基因导入ES细胞，在添加了玉米素的培养液中培养，用聚合酶链式反应检测克隆的表达。细胞在不添加人白血病抑制因子的培养液中培养，分化形成类胚体。类胚体的一些细胞开始自发跳动。有表达绿色荧光蛋白的细胞存在，但不能检测到足够数量的转…基因的细胞。更多采用流式细胞仪分析。我们交替地进行了嘌呤霉素的筛选，但该选择不能有效地检测到足够数量的拍动细胞。因为没有检测到分化为心肌细胞的特异性主控基因。从培养的分化ES细胞中分离出心肌表型细胞似乎是一种很有前途的策略。然而，转导的基因在ES细胞中出人意料地没有很好地激活。该基因可能通过未知的基因失活机制被关闭。这可能是转导启动子基因的甲基化，被推测为失活的可能机制之一。为了评价细胞移植的效果，我们采用了冷冻损伤模型。用低温探头损伤Fisher大鼠心肌，用超声晶体探头分析左心室局部运动情况。然而，该测量技术在检测心功能下降方面的准确性受到限制。较少

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