诱导小鼠颊黏膜成纤维细胞形成诱导性多潜能干细胞  

Generation of induced pluripotent stem cells from mouse buccal mucosa fibroblasts

作　　者：尹映竹 梁国斌[1] 刀力 张新春[1] 覃峰[1] 杨凌[1] 

机构地区：[1]中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室,广州510055

出　　处：《中华口腔医学研究杂志（电子版）》2017年第6期354-361,共8页Chinese Journal of Stomatological Research(Electronic Edition)

基　　金：国家自然科学基金(青年科学基金项目,81100775);广东省自然科学基金(2017A030313863)

摘　　要：目的研究小鼠颊黏膜成纤维细胞(BF)重编程形成诱导性多潜能干细胞(iPSC)的诱导方法。方法应用携带有Oct4、Sox2和Klf4基因的逆转录病毒诱导小鼠BF重编程形成iPSC。观察iPSC克隆形态;进行碱性磷酸酶(ALP)染色、核型分析;实时荧光定量聚合酶链反应(PCR)及细胞免疫荧光术检测内/外源性多能基因和干细胞标志物的表达;体外、体内分化实验检测iPSC多向分化潜能。结果 iPSC呈典型胚胎干细胞(ESC)样克隆生长,边界清晰;细胞ALP染色阳性,核型正常。内源性Dppa3、Nanog、Rex1、Sox2、Klf4、Oct4、c-Myc基因表达量与ESC接近,未检测到外源性Sox2、Klf4、Oct4、c-Myc基因表达。干细胞标志物Oct4、Sox2和Nanog表达呈阳性。在体外,iPSC可被诱导向成骨、成脂、成软骨方向分化;体内可分化成多种组织,形成畸胎瘤。结论成功诱导BF重编程形成iPSC,为牙再生医学相关研究提供种子细胞来源。Objective To generate induced pluripotent stem cells（iPSCs）from mouse buccal mucosa fibroblasts（BFs）by genetic reprogramming. Methods iPSCs were established from mouse BFs via retroviral gene transfer with three reprogramming factors（Oct4,Sox2,and Klf4）. The properties of iPSCs were characterized by alkaline phosphatase staining assay,karyotype analysis,real - time fluorescence quantitative polymerase chain reaction（qRT-PCR）,immunofluorescence and bisulfite genomic sequencing. In vitro and in vivo studies were also performed to test their differentiation and pluripotent capability. Results The resulting iPSCs were found to resemble mouse embryonic stem cells（ESCs）with similar clone growth and high positive stain of alkaline phosphatase. They had normal karyotype,and expressed high levels of ESCs-like genes（Dppa3,Nanog,Rex1,Sox2,Klf4,Oct4 and c-Myc）and protein markers （Oct4,Sox2,and Nanog）. Bisulfite sequencing demonstrated that the methylation of the endogenous Oct4 and Nanog promoters in iPSCs and the control ESCs clones was low or absent,while a high methylation level was found in mouse embryonic fibroblasts（MEFs）. By in vitro induction,iPSCs could produce mineralized nodules,form adipocyte-like cells with lipid droplets,and change to chondrocytes. Moreover, they can differentiate into teratomas composed of tissues of the three germ layers in vivo. Conclusions iPSCs were successfully established from the mouse BFs with ESCs-like properties and their pluripotency was verified. These iPSCs may serve as seed cells for tooth regeneration.

关 键 词：诱导多功能干细胞 成纤维细胞 细胞重编程 再生 

分 类 号：R78[医药卫生—口腔医学]