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目的探讨小鼠胚胎干细胞(ES)分化的心肌细胞移植对SD大鼠心肌梗死后左心室重构及心功能的影响。方法采用悬滴-悬浮培养法诱导ES分化形成拟胚体,其中包含有大量心肌细胞。将雄性SD大鼠冠状动脉左前降支结扎,制成急性心肌梗死模型,1周后再次开胸,实验组于梗死区域注入心肌细胞悬液150μl(含细胞5×10~5个),梗死对照组于梗死区域注入等体积细胞培养液,另设假手术组,不结扎冠状动脉。心肌梗死后1周和细胞移植后4周,采用超声心动图评价大鼠左心室形态和功能,用免疫荧光技术检测移植细胞的存活及其表型。结果免疫荧光技术检测到分化的心肌细胞表达特异性转录因子α辅肌动蛋白、α肌球蛋白重链和肌钙蛋白Ⅰ;逆转录聚合酶链反应检测到有心肌特异性转录因子Nkx-2.5、GATA-4及α肌球蛋白重链基因的表达;膜片钳记录显示所得到的分化的心肌细胞包括窦房结样起搏细胞、心房样细胞和心室样细胞。细胞移植后4周,与梗死对照组相比,实验组左心室收缩末期容积和舒张末期容积均显著缩小,左心室前壁舒张末期厚度明显增加(P<0.01),左室长轴缩短率显著升高(P<0.01)。心脏组织冰冻切片可见4,6-联脒-2-苯基吲哚标记的呈蓝色荧光的移植细胞核,移植细胞表达肌钙蛋白Ⅰ,移植细胞与受者的心肌细胞间有连接蛋白- 43的表达。实验组中有2只(12.5%,2/16)出现了心脏畸胎瘤。结论由胚胎干细胞分化的心肌细胞移植可逆转心肌梗死大鼠左心室重构,改善左心室功能。
Objective To investigate the effects of embryonic stem cell (ES) -induced cardiomyocyte transplantation on left ventricular remodeling and cardiac function after myocardial infarction in SD rats. Methods ESCs were induced by suspension-suspension culture to form embryoid bodies, which contained a large number of cardiomyocytes. The left anterior descending coronary artery of male Sprague-Dawley rats were ligated to make a model of acute myocardial infarction. Thoracotomy was performed one week later. The experimental group was infused with 150μl of cardiomyocyte suspension (5 × 10 ~ 5 cells) In the infarct area, an equal volume of cell culture fluid was injected into the infarct area. A sham-operated group was also set up, and the coronary artery was not ligated. Left ventricular morphology and function were evaluated by echocardiography 1 week after myocardial infarction and 4 weeks after cell transplantation. The survival and phenotype of transplanted cells were detected by immunofluorescence. Results The expression of specific transcription factors α-actinin, α-myosin heavy chain and troponin I in differentiated cardiomyocytes were detected by immunofluorescence technique. The myocardial specific transcription factor Nkx-2.5 was detected by reverse transcription polymerase chain reaction , GATA-4 and α-myosin heavy chain genes; patch-clamp recordings showed that the resulting differentiated cardiomyocytes included sinoatrial-like pacemaker cells, atrial-like cells and ventricular-like cells. Compared with infarction control group, left ventricular end-systolic volume and end-diastolic volume of experimental group decreased significantly at 4 weeks after cell transplantation, and left ventricular anterior wall diastolic thickness increased significantly (P <0.01), shortening of left ventricular long axis was significant Increased (P <0.01). Frozen sections of the heart tissue showed a blue fluorescent transplanted nucleus labeled with 4,6-diamidino-2-phenylindole, and the transplanted cells expressed troponin I with connexin 43 between the transplanted cells and the recipient’s cardiomyocytes expression. Two (12.5%, 2/16) of the experimental group developed cardiac teratoma. Conclusion Cardiomyocytes differentiated by embryonic stem cells can reverse left ventricular remodeling and improve left ventricular function in rats with myocardial infarction.