体内外获取心脏起搏细胞的方法学探讨  

The methodology of harvesting cardiac pacemaker cells by vivo and vitro

作　　者：张进[1] 魏飞宇 王礼琳[1] 丁立群[1] 张曦[1] 匡晓晖 李颖[1] 白鹤群 范洁[1] 

机构地区：[1]云南省第一人民医院心内科,昆明650032

出　　处：《临床心血管病杂志》2017年第4期367-370,共4页Journal of Clinical Cardiology

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)

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(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 establish a new method of human iPS cells differentiate into cardiac pacemaker cells in vitro and separation of rabbit sinoatrial node tissue by accurate anatomy positioning in vivo.Method：Inducing of human iPS cells differentiation into pacemaker cells by Cellapy method,macroscopic was used to observate the pacemaker cell morphology and pacing rate.Immunofluorescence was used to detect expression of HCN4 positive ratio of the differentiation of pacemaker cells.Meanwhile,rabbit sinoatrial node tissue was separate by the precise anatomical,qRT-PCR and immune blotting was performed to detect HCN4 relative mRNA and protein expression level.Result：Human iPS cells was successfully induced differentiate into spontaneously beating pacemaker cells by Cellapy method.The morphological forms of pacemaker cell were mainly fusiformis cells and the beating rate was about（70.60±2.71）beats per minute;immunofluorescence showed that the HCN4 positive cells was accounted for（9.42±1.57%）in the induced differentiation of cells;qRT-PCR and blotting results showed that the mRNA and protein of HCN4 expression was significantly higher in sinoatrial node tissue than the atrial and ventricular myocytes（P〈0.01）.Conclusion：The present study demonstrated that human iPS cells was induced differentiate into cardiac pacemarker cells by Cellapy method and accurate separation of rabbit sinoatrial node were effective methods to harvesting cardiac pacemaker cells,which provides an important cell resources for studying molecular mechanism and treatment of sick sinus syndrome.

关 键 词：IPS细胞 起搏细胞 窦房结组织 HCN4 病态窦房结综合征 方法学 

分 类 号：R541.7[医药卫生—心血管疾病]