体内外获取心脏起搏细胞的方法学探讨

发布时间：2019-04-02 16:06

【摘要】：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(P0.01)。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。
[Abstract]:Aim: to induce human induced pluripotent stem cells (induced pluripotent stem cell,iPS) to differentiate into cardiac pacing cells in vitro. To obtain rabbit sinoatrial node tissue by anatomical localization in vivo, and then to obtain cardiac pacing cells directly. Methods: human iPS cells were induced to differentiate into cardiomyocytes and cardiac pacing cells by Cellapy method in vitro. The morphology and frequency of pacemaker cells were observed by naked eyes. The HCN4 positive ratio of specific markers in human iPS-derived cardiac pacing cells was detected by immunofluorescence. The expression of HCN4mRNA and protein in rabbit cardiac pacing cells were detected by qRT-PCR and Western blot after the rabbit cardiac pacing cells were accurately located in vivo. Results: human iPS cells were successfully induced to differentiate into pacemaker cells by Cellapy method. The pacemaker frequency of pacemaker cells was (70.60 卤2.71) beats / min, and the morphology of pacemaker cells was spindle type. Immunofluorescence showed (9.42 卤1.57)% of HCN4 protein positive cells in human iPS-derived cardiac pacing cells. At the same time, the results of qRT-PCR and Western blot showed that the expression of HCN4 in pacemaker cells of rabbit sinoatrial node was significantly higher than that of atrial muscle and ventricular muscle (P0.01). Conclusion: Cellapy induction in vitro and anatomical localization in vivo are two effective methods for obtaining cardiac pacing cells. This provides a reliable source of cells for the study of pathogenesis of sick sinus syndrome, and provides transplantation cells for the treatment of sick sinus syndrome.
【作者单位】：云南省第一人民医院心内科;
【基金】：国家自然科学基金项目(No:81260038,81360039) 云南省心律失常诊治研究中心研究基金项目(No:2014NS260,2014NS259)
【分类号】：R541.7

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