小鼠诱导多能干细胞体外分化为心肌细胞的实验研究  

Research of Mouse Induced Pluripotent Stem Cells Differentiate into Cardiomyocytes in vitro

作　　者：熊挺淋 张丽莎[1,2] 刘涛 王浩宇[1,2] 赵超美 周振宇[1,2] 赵霞 杨梁[3] 

机构地区：[1]南充市中心医院心内科 [2]川北医学院第二临床医学院,南充637000 [3]重庆市神经病学重点实验室,重庆400016

出　　处：《中国细胞生物学学报》2013年第6期810-815,共6页Chinese Journal of Cell Biology

基　　金：重庆市卫生局医学科学技术研究重点项目(批准号:2009-1-51)资助的课题~~

摘　　要：观察血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)和维生素C对小鼠诱导多能干细胞(induced pluripotent stem cells,iPSCs)体外分化为心肌细胞的诱导作用。采用直接悬浮培养法使iPSCs形成拟胚体(embryoid bodies,EBs),分别以VEGF和维生素C作为诱导剂,自然分化作为阴性对照组,加入1%二甲亚砜诱导剂为阳性对照组。倒置显微镜下观察细胞生长情况,记录跳动的拟胚体出现的时间和数目,计算心肌细胞分化率,细胞免疫荧光检测心肌特异性蛋白cTnT的表达,RT-PCR检测心肌特异性基因β-MHC mRNA的表达。在LIF条件下,iPSCs在饲养层上成集落状生长;未分化的iPSCs中Oct-4及AKP呈阳性表达;与自然分化组相比,二甲亚砜、VEGF和维生素C均能提高iPSCs的心肌细胞分化效率(P<0.05);分化的心肌细胞可自发搏动,同时分化细胞中表达心肌特异性蛋白cTnT以及心肌特异性基因β-MHC。VEGF和维生素C可以促进iPSCs向心肌细胞分化。To observe the effect of vascular endothelial growth factor （VEGF） and vitamin C on the cardiomyocytes differentiation of mouse induced pturipotent stem cells （iPSCs） in vitro. Embryoid bodies （EBs） was generated from iPSCs by direct suspendsion culture, with VEGF and vitamin C as inducers, natural differentiation without any inducer was negative control group and 1% dimethyl sulfoxide was added as positive control group. Cells were observed under inverted microscope, and the time of beating embryonic bodies appeared and numbers were recorded and the differentiation rate of cardiomyocyte was calculated, cTnT expression of cardiac-specific protein was detected by immunofluorescence and β-MItC mRNA expression of cardiac-specific gene were detected by RT-PCR. iPSCs showed colony-like growth on feeder layer cells in the presence of leukemia inhibitory factor （LIF）. Undifferentiated iPSCs expressed Oct-4 and alkaline phosphatase （AKP）. Compared with the natural differentiation group, dimethyl sulfoxide, VEGF and vitamin C all improved cardiomyocytes differentiation rate of iPSCs （P〈0.05）. Cardiomyocytes derived from iPSCs beated spontaneously and expressed cardiac-specific protein cTnT and cardiac-specific gene β-MHC. VEGF and vitamin C can promote iPSCs into cardiomyocytes.

关 键 词：诱导多能干细胞 血管内皮细胞生长因子 维生素C 诱导分化 心肌细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]