【摘要】 目的探究通过阶段性调控经典Wnt信号通路促使人诱导多潜能干细胞向心肌细胞分化。方法采用阶段性激活或抑制Wnt信号通路的方案使人尿路上皮细胞来源的多潜能干细胞（U1细胞系）定向分化为心肌细胞,在分化第14天,采用流式细胞术检测U1细胞向心肌细胞分化的效率,利用免疫细胞化学检测心肌细胞肌钙蛋白表达,通过激光扫描共聚焦荧光显微镜检测人诱导多潜能干细胞来源的心肌细胞（hiPSC-CMs）的Ca²⁺活动,利用全细胞膜片钳技术记录hiPSC-CMs的电生理特性。结果 U1细胞系向hiPSC-CMs分化的效率为（88±3）%;全细胞膜片钳技术记录的动作电位特征显示hiPSC-CMs可分为心室肌样细胞、心房肌样细胞和窦房结样细胞,其中主要是心室肌样细胞。窦房结样细胞的比例、动作电位时程、动作电位幅度及最大去极化速率均明显小于心室肌样细胞和心房肌样细胞。结论阶段性调控Wnt信号通路能促使hiPSCs定向分化为hiPSC-CMs,并且hiPSC-CMs具有哺乳动物心肌细胞相似的电生理特征。更多还原

【Abstract】 Objective To promote the differentiation of human induced pluripotent stem cells(hiPSCs) into cardiomyocytes through the manipulation of the canonical Wnt signaling pathway. Methods hiPSCs(U1 cell lines) derived from undifferentiated healthy adult urothelium cells were differentiated into cardiomyocytes by staged activation or inhibition of the Wnt signaling pathway. On the 14th day of differentiation,flow cytometry was used to detect the differentiation efficiency of U1 cell lines into cardiomyocytes. Immunocytochemistry was utilized to detect cardiac troponin T expression in cardiomyocytes. The calcium activity of hiPSC-CMs was recorded by laser scanning confocal fluorescence microscopy. The electrophysiological characteristics of hiPSC-CMs were detected by whole cell patch clamp techniques. Results The efficiency of U1 cell line differentiation to hiPSC-CMs was(88±3) %. Action potential characteristics recorded by whole-cell patchclamp techniques demonstrated that hiPSC-CMs could be divided into ventricular-like cardiomyocytes,atrial-like cardiomyocytes and sinoatrial node-like pacemaker cells,but mainly ventricular-like cardiomyocytes. The proportion,action potential duration,action potential amplitude and maximum depolarization rate of sinoatrial node-like pacemaker cells were significantly lower than those of ventricular-like cardiomyocytes and atrial-like cardiomyocytes. Conclusion The phased modulation of Wnt signaling pathway could induce hiPSCs to differentiate into hiPSC-CMs with high differentiation efficiency,and hiPSC-CMs have similar electrophysiological characteristics with mammalian cardiomyocytes.更多还原