Embryonic stem cell (ES cell)-derived cardiomyocyte transplantation into the infarcted myocardium

本研究的目的是研究胚胎干细胞（ES细胞）来源的心肌细胞移植到梗死心肌中的存活情况。在体外研究中，将携带增强绿色荧光蛋白（EGFP）的未分化小鼠胚胎干细胞挂滴培养7 d，然后镀于培养皿上，然后解剖跳动团，用于后续分析和移植。通过心脏特异性基因的逆转录聚合酶链反应、动作电位记录和肌球蛋白染色的免疫染色，这些细胞被鉴定为心肌细胞。体外研究结果显示，ES细胞来源的心肌细胞具有肌球蛋白轻链-2v和α -肌球蛋白重链基因表达，动作电位与窦结细胞和心室肌细胞的动作电位相似，肌球蛋白染色阳性。采用左冠状动脉结扎法制备大鼠心肌梗死模型。在梗死心肌和正常心肌的交界区注射跳动团。移植后10天和30天，观察EGFP的表达、苏木精-伊红染色和肌球蛋白免疫染色。在体内研究中，移植后检测到表达egfp的细胞，其中部分细胞肌球蛋白阳性。未见淋巴细胞浸润。这些结果表明，胚胎干细胞在体外分化为心肌细胞，胚胎干细胞衍生的心肌细胞可以移植到梗死心肌中并在体内存活。

The purpose of this study was to investigate the survival of embryonic stem cell (ES cell)-derived cardiomyocytes transplanted into the infarcted myocardium.In in vitro study, undifferentiated mouse ES cells carrying the enhanced green fluorescent protein (EGFP) were cultured in hanging drops for 7 days and plated onto dishes, and then beating clusters were dissected and used for the following analyses and transplantation. These cells were identified as cardiomyocytes by reverse transcription-polymerase chain reaction of cardiac-specific genes, recording of action potential, and immunostaining of sarcomeric myosin staining. As a result of in vitro study, ES cell-derived cardiomyocytes were shown to have myosin light chain-2v and alpha-myosin heavy chain genes expression, action potential like one from a sinus nodal cell and a ventricular myocyte, and positive for sarcomeric myosin staining.In in vivo study, a myocardial infarction rat model was made by the ligation of the left coronary artery. Beating clusters were injected into the border zone between the infarcted and normal myocardium. Ten and 30 days after transplantation EGFP expression, hematoxylin-eosin staining and immunostaining of sarcomeric myosin were investigated. As a result of in vivo study, EGFP-expressed cells were detected after transplantation, and a few of them were positive for sarcomeric myosin. No lymphocytic infiltration was observed.These results suggested that ES cells differentiated into cardiomyocytes in vitro and ES cell-derived cardiomyocytes could be transplanted into the infarcted myocardium and survive in vivo.