人诱导性多能干细胞的培养及鉴定  

Culture and identification of human induced pluripotent stem cells

作　　者：冯年花[1,2] 谢安[1] 娄远蕾[1] 阮琼芳[1] 郭菲[1] 吴珏[1,2] 邓志锋[3] 汪泱[1] 

机构地区：[1]南昌大学泌尿外科研究所 [2]南昌大学研究生院医学部 [3]南昌大学第二附属医院神经外科,江西南昌330006

出　　处：《实验与检验医学》2010年第1期6-8,15,共4页Experimental and Laboratory Medicine

基　　金：国家自然科学基金(30960385);江西省自然科学基金(2007GZY1470)

摘　　要：目的建立稳定的人诱导性多能干细胞(iPS细胞)培养体系。方法取第3～5代的小鼠胚胎成纤维细胞,丝裂霉素C处理后用作饲养层。人iPS细胞接种于饲养层上生长,胶原酶消化或机械法传代。倒置显微镜下观察iPS细胞生长状态;观察拟胚体形成能力;RT-PCR检测iPS细胞多能性基因的表达情况。结果(1)小鼠胚胎成纤维细胞(MEF)为贴壁生长细胞,呈梭形或多角形。细胞增殖旺盛。(2)生长在饲养层上的人iPS细胞呈典型的克隆状生长。克隆呈圆形或椭圆形,边界清晰。克隆内细胞排列紧密,细胞体积小,核大,细胞核/质比高。RT-PCR结果显示人iPS细胞强表达多能性基因Oct4,Nanog,Sox2;去除饲养层后悬浮培养能形成拟胚体(EB)。结论本实验的培养体系适合人iPS细胞的培养,人iPS细胞能稳定增殖,保持自我更新及分化潜能。Objective To establish a stable culture system of induced pluripotent stem cells（iPS）. Methods Mouse embryonic fibroblast cells of 3-5th passages were treated by mitomycin C and prepared as feeder layers. iPS clones were plated on feeder layers and cells were observed under invert microscope, iPS clones was passaged by eollagenase digestion or mechanical method. Total mRNA were extracted and expression of pluripotentassociated genes were detected by RT-RCR. Results （1） Mouse embryonic fibroblast cells were tightly adherent to culture dish and showed a spindle or polygon shape. （2） iPS cells were maintained on inactivated feeder layer and formed condensed clones with clear borders. Cells in clones appeared high nucleus/cytoplasm ratio and predominant nuclei. All iPS cells strongly expressed Oct4, Nanog and Sox2. When feeder cells were removed, iPS cells formed embryoid bodies in suspension condition. Conclusion Human iPS cells proliferated stablely and kept high self-renewal and differentiation potency in our lab culture system, so the culture system is suitable for the long term subculture of human iPS cells.

关 键 词：人诱导性多能干细胞 细胞培养 饲养层 

分 类 号：R446.113[医药卫生—诊断学] Q813.11[医药卫生—临床医学]