基于iPSC诱导分化具有电生理活动心肌细胞方法的研究  

Generation of cardiomyocytes from induced pluripotent stem cells

作　　者：李弘夏[1] 刘涛燕[1] 吴福建[1] 兰峰[1] 

机构地区：[1]首都医科大学附属北京安贞医院-北京市心肺血管疾病研究所血管生物研究室,100029

出　　处：《心肺血管病杂志》2016年第11期896-899,共4页Journal of Cardiovascular and Pulmonary Diseases

基　　金：优秀青年科学基金项目(81422003);国家自然科学基金面上项目(81570215)

摘　　要：目的:通过诱导多潜能干细胞i PSC体外定向分化为心肌细胞的方法,已经成为干细胞和心血管领域研究的有效工具,也为临床治疗带来新希望。本文依据胚胎发育的分子调控机制,探索建立体外定向分化心肌的方法。方法:使用干细胞培养基培养i PSCs生长至80%,换成含6μmol/L Chir99021和25μg/L Activin A的基础培养基培养48h,基础培养基为含有2%的B27、64μg/L l-ascorbic acid2-phosphate的RPMI 1640培养基,换为含2μM Wnt-C59的WNT信号抑制剂培养基培养48h,最后换为维持培养基继续培养,每两天换液,约第8天可见心肌开始搏动。结果:i PSC在向成熟心肌分化时,显微镜下可观察到明显的肌丝肌节结构,具有心肌特异性标志物TNNT 2,MLC 2a的强阳性表达,并表现出自发跳动的电生理活动。结论:该方法可以成功诱导i PSC高效分化为具有电生理功能的心肌细胞。Objective： The recently developed method inducing pluripotent stem cells（ induced pluripotent stem cells,i PSCs） to differentiation into cardiomyocytes in vitro has become an effective tool in the field of cardiovascular and stem cell research,but also brings new hope for clinical treatment. Based on the mechanism of molecular regulation of embryonic development,we explored the method of cardiac differentiation in this study. Methods： i PSCs clones were routinely maintained in stem cell culture medium,at which time they reached ~80% confluence,medium was changed to the basal differentiation medium,consisting of 2% B27,64μg / L Lascorbic acid 2-phosphate in RPMI 1640 culture medium. For day0 to day2,medium was supplemented with 6μM CHIR99021 and 25μg /L Activin A. On day 2,medium was changed to the basal differentiation medium supplemented with 2 μM Wnt-C59. Medium was changed on day 4 and every other day for the basal differentiation medium. Contracting cells were noted from day 8. Results： i PSCs derived cardiomyocytes expressed cardiac markers of TNNT 2 and MLC 2a,had obvious sarcomere structure and characteration of spontaneous beating.Conclusion： this protocol successfully and efficiently produced contractile cardiomyocytes from iPSCs.

关 键 词：诱导多潜能干细胞 分化 心肌细胞 

分 类 号：R54[医药卫生—心血管疾病]