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目的:体外诱导人诱导性多能干细胞(induced pluripotent stem cell,iPS)分化为心脏起搏细胞,体内解剖精确定位获得兔窦房结组织,进而直接获得心脏起搏细胞。方法:体外Cellapy法高效诱导人iPS细胞分化为心肌细胞和心脏起搏细胞,肉眼观察心脏起搏细胞形态及起搏频率,免疫荧光检测人iPS源性心脏起搏细胞中特异性标志物HCN4阳性比例。体内解剖精确定位获得兔心脏起搏细胞后,应用qRT-PCR及免疫印迹法检测兔心脏起搏细胞HCN4mRNA及蛋白表达水平。结果:运用Cellapy方法成功将人iPS细胞诱导分化为具有自发跳动特性的心脏起搏细胞,显微摄像记录显示心脏起搏细胞搏动频率为(70.60±2.71)次/min,且细胞形态以梭型为主;免疫荧光显示人iPS源性心脏起搏细胞中HCN4蛋白阳性细胞占(9.42±1.57)%。同时,qRT-PCR及Western blot结果显示兔窦房结组织中心脏起搏细胞HCN4表达明显高于心房肌和心室肌(P<0.01)。结论:体外Cellapy诱导法和动物体内解剖精确定位是获得心脏起搏细胞的两种有效方法。这为研究病态窦房结综合征的发病机制提供了可靠的细胞来源,同时为病态窦房结综合征的治疗提供了移植细胞。
OBJECTIVE: To induce induced pluripotent stem cells (iPS) to differentiate into cardiac pacemaker cells in vitro and obtain the sino-atrial node tissue by anatomical and precise positioning in vivo, and to obtain cardiac pacemaker cells directly. Methods: Human iPS cells were induced to differentiate into cardiomyocytes and cardiac pacemaker cells by Cellapy method in vitro. The shape and pace of cardiac pacemaker cells were observed by naked eyes. Immunofluorescence was used to detect the positive expression of HCN4, a specific marker in human iPS-derived cardiac pacemaker cells proportion. Accurate localization of the rabbit cardiac pacemaker cells in vivo, qRT-PCR and Western blotting were used to detect HCN4 mRNA and protein expression in rabbit cardiac pacemaker cells. Results: The celiac cells were successfully differentiated into spontaneous beating cardiac pacing cells by Cellapy method. The frequency of pulsating pacemaker cells was (70.60 ± 2.71) / min. . The expression of HCN4 positive cells in human iPS-derived cardiac pacemaker cells was (9.42 ± 1.57)%. Meanwhile, qRT-PCR and Western blot showed that the expression of HCN4 in cardiac pacemaker cells was significantly higher than that in atria and ventricular muscles (P <0.01). CONCLUSION: In vitro Cellapy induction and accurate dissection in animals are two effective methods for obtaining cardiac pacemaker cells. This provides a reliable source of cells for the study of the pathogenesis of sick sinus syndrome and at the same time provides a transplanted cell for the treatment of sick sinus syndrome.