AIM:To study the direct reprogramming method of mouse embryonic fibroblasts (MEFs) conver-ted into induced neural stem cells (iNSCs).METHODS:Sox2-infected MEFs were cultured in NSCs culture medium for 10 d.Subsequently , repeated suspension and adherent culture were performed for 3 times for the purification of iNSCs .The iNSCs were cultured in suspension medium .Real-time PCR was used to detect the expression of neural stem cell marker genes and pluripotent marker gene .In vivo, iNSCs were microinjected into the mouse cerebral cortex .Immunofluorescence was performed to detect the expression of neural stem cell , neuron, oligodendrocyte and astrocyte markers in vitro and vivo. RESULTS:A variety of neural stem cell marker gene expression was significantly increased in iNSCs detected by real -time PCR.Immunofluorescence confirmed that iNSCs expressed nestin and differentiated into neurons , oligodendrocytes and as-trocytes in vitro and vivo.CONCLUSION:Sox2 is sufficient to trigger the direct reprogramming from MEFs to iNSCs .iN-SCs have the ability of self-renew and 3 differentiation potentials in vivo and vitro.iNSCs are the suitable seed cells of SCI .%目的：探讨Sox2诱导小鼠胚胎成纤维细胞（ mouse embryonic fibroblasts ， MEFs）直接重编程为神经干细胞的方法，为诱导神经干细胞（induced neural stem cells ， iNSCs）作为种子细胞治疗脊髓损伤（spinal cord injury， SCI）奠定实验基础。方法：逆转录病毒感染Sox2转录因子的MEFs在神经干细胞培养基中贴壁培养10 d。随后，悬浮、贴壁反复循环培养3次。 Real-time PCR检测神经干细胞标志基因和多潜能标志基因的表达。 iNSCs在分化培养基中贴壁培养7～14 d，免疫荧光分别检测神经干细胞、神经元、星形胶质细胞和少突胶质细胞的标志物nes-tin、MAP2、GFAP和MBP的表达。将iNSCs显微注射至小鼠大脑皮质，7～14 d后免疫荧光检测神经细胞标志物nestin、MAP2、GFAP和MBP的表达。结果：Real-time PCR显示iNSCs的多种神经干细胞标志基因nestin、Blbp、Pax6和zbtb16表达较诱导前显著增高（P＜0.05），且iNSCs不表达多潜能相关基因Nanog、Oct4和zfp42。免疫荧光显示iNSCs高表达神经干细胞标志物nestin。免疫荧光同时表明iNSCs可在体内外存活并分化为神经元、星形胶质细胞和少突胶质细胞。结论：Sox2可以诱导MEFs直接重编程为神经干细胞。 iNSCs具有自我更新的能力，且在体内外都具有三向分化潜能，可作为修复SCI合适的种子细胞。
展开▼
