Cell transplantation to myocadial infarction area

本研究的目的是评估细胞移植到心肌梗死模型的啮齿动物的应用。培养胚胎干细胞并诱导其无意中分化，显示出表达心肌表型的细胞。我们推测胚胎干细胞分化为心肌细胞表达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和嘌呤霉素抗性基因的cDNA组成的表达基因，并将该基因转染ES细胞。将转染该基因的ES细胞培养在添加有zeoncin的培养基中，并通过PCR确定转染的克隆。细胞在不添加人白血病抑制因子的培养基中培养并分化形成胚样体。胚状体的一些细胞开始自发地跳动。存在表达EGFP的细胞，但我们不能检测到足够数量的基因转染的细胞 关于我们 即通过流式细胞术分析。我们进行了嘌呤霉素选择，但选择是不是有效地检测到足够数量的跳动细胞。由于未检测到分化为心肌细胞的特异性主基因。从体外培养的胚胎干细胞中分离心肌表型细胞是一种很有前途的方法。然而，转染的基因在ES细胞中并没有很好地激活。转染的基因似乎被未知的基因失活机制所关闭。这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。为了评价细胞移植的有效性，我们使用冷冻损伤模型。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。然而，测量技术的准确性对于检测心脏功能的降低是有限的。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