人诱导多能干细胞定向分化为间充质干细胞的方法研究  

Study on method of directional differentiation of human induced pluripotent stem cells towards mesenchymal stem cells

作　　者：胡国文[1] 李青[2] 牛鑫[2] 胡斌[2] 刘鹃[1] 郎海丽[3] 沈晓黎[1] 汪泱[2] 邓志锋[1,4] 

机构地区：[1]南昌大学第二附属医院神经外科,南昌330006 [2]上海交通大学附属第六人民医院四肢显微外科研究所,上海200233 [3]南昌大学第二附属医院麻醉科,南昌330006 [4]上海交通大学附属第六人民医院神经外科,上海200233

出　　处：《上海交通大学学报（医学版）》2014年第10期1428-1434,共7页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(81272170;81472152)~~

摘　　要：目的研究一种高效、简捷的直接将人诱导多能干细胞(hiPS)诱导分化为间充质干细胞(MSCs)的方法。方法用MSCs诱导培养基将iPS诱导为MSCs样细胞,倒置显微镜下观察诱导过程中细胞形态的变化,流式细胞术检测iPS及iPS-MSCs表面标志物的表达,RT-PCR检测诱导过程中细胞内NANOG、OCT-4、MSX-1等干性基因表达水平,成骨、成脂、成软骨诱导鉴定其三系分化能力。结果诱导后iPS逐渐向外生长,P4代iPS-MSCs逐渐变为长梭形;CD29、CD44、CD73、CD90、CD105在iPS-MSCs中表达阳性,而CD34、CD45、HLADR则为阴性;iPS在诱导过程中NANOG和OCT-4基因表达逐渐降低,iPS-MSCs不表达NANOG和OCT-4基因,MSX-1基因在诱导第4天即高表达并维持在高水平;iPS-MSCs具有成骨、成脂、成软骨能力。结论成功建立一种高效、简捷的直接将iPS诱导为功能性的MSCs样细胞的方法,为iPS-MSCs进一步的研究与应用提供了技术基础。Objective To develop an efficient and simple method to induce the differentiation of human induced pluripotent stem cells（ hiPS） towards mesenchymal stem cells（ MSCs）. Methods MSCs induction medium was used to induce iPS towards MSCs.The cell morphology was observed under the inverted microscope during the course of induction.The expressions of surface markers of iPS and iPS-MSCs were detected by the flow cytometry. The expressions of stem ness genes such as NANOG, OCT-4, and MSX-1 in cells during the course of differentiation were detected by the RT-PCR. The osteogenesis, adipogenesis, and chondrogenesis were used to identify their tri-lineages differentiation pluripotency. Results The iPS gradually grew outward after being induced and M SC s gradually became fusiform in Passage 4.The iPS-MSCs were positive to CD29, CD44,CD73, CD90, and CD105 and negative to CD34, CD45, and HLA-DR. The expressions of NANOG and OCT-4 gradually decreased during the course of differentiation and NANOG and OCT-4 were not expressed in iPS-MSCs. The expression of MSX-1 increased to a high level after being induced for 4 d and maintained afterwards. The iPS-MSCs had abilities of osteogenesis, adipogenesis, and chondrogenesis. Conclusion An efficient and simple method is developed for directly inducing iPS towards functional iPS-MSCs,which provides the technical basis for further research and application of iPS-MSCs.

关 键 词：诱导多能干细胞 间充质干细胞 细胞分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]