皮肤源性再程序化神经干细胞的制备以及定向分化为神经细胞的体外研究

The study on preparation of reprogrammed neural stem cells derived from skin and neural differentiation in vitro

目的:研究制备皮肤源性再程序化神经干细胞以及诱导其定向分化为神经细胞的可行性。方法:体外培养大鼠皮肤来源前体细胞(skin-derived precursors,SKPs)并纯化、鉴定,用含有绿色荧光蛋白(GFP)基因的慢病毒载体系统作为基因转染工具,将第3代SKPs分3组:A组为慢病毒载体介导neurogenin2(Ngn2)基因转染的SKPs;B组为空载体慢病毒转染的SKPs;C组为未进行慢病毒介导基因转染的SKPs;7天后加入无生长因子含10%胎牛血清的诱导培养基诱导7天。光镜下观察比较各组细胞形态变化以及免疫荧光镜下研究各组SKPs诱导后定向分化为神经细胞的差异。结果:A组SKPs转基因后到第7天绿色荧光达到高峰,并可持续稳定表达绿色荧光至第8代;诱导7天后绝大部分细胞类似神经细胞,胞体呈梭形或椭圆形,有多个突起伸出;免疫荧光结果显示90.98%细胞表达微管相关蛋白-2(MAP-2),62.23%细胞表达神经元特异性蛋白NeuN。B组SKPs在空病毒转染7天以后有绿色荧光;诱导7天以后大多数细胞较诱导前无明显变化;免疫荧光结果显示,有34.35%的细胞表达MAP-2,无神经元特异性蛋白NeuN的表达。C组SKPs无绿色荧光;诱导7天以后大多数细胞较诱导前无明显变化;免疫荧光结果显示,29.54%的细胞表达MAP-2,无神经元特异性蛋白NeuN的表达。结论:慢病毒介导Ngn2基因转染SKPs可以制备出皮肤源性再程序化的神经干细胞,并提高其定向分化为神经细胞的能力。

Objective：To evaluate the feasibility of preparation of reprogrammed neural stem cells derived from skin and neural differentiation in vitro.Methods：Skin-drived precursors（SKPs） were isolated and purified from rat skin tissue,identified and seeded at passage 3 in 12-well culture plates.SKPs were divided into three groups：group A was transfected with neurogenin2 via GFP-plentivirus;group B was transfected with GFP-plentivirus;group C was cultured without transfection.All groups were induced by common inducer for a week after 7 days of transfection.The morphology of induced SKPs was observed by microscope,and the markers of neural cells（MAP-2 and NeuN） were detected by immunocytochemistry.Results：The green fluorescence of SKPs in group A reached the peak on the 7th day after transfection and existed until the 8th passage.The shapes of SKPs were fusiform or ellipse,and most SKPs express several apophyses after inducement.Immunofluorescence demonstrated that 90.98% of the SKPs expressed MAP-2 and 62.23% of the SKPs expressed NeuN.SKPs in group B expressed green fluorescence and had no significant typical morphological changes after inducement.Immunofluorescence demonstrated that 34.35% of the SKPs epressed Map-2 and no SKPs expressed NeuN;SKPs in group C did not express green fluorescence and had no significant typical morphological changes after inducement.Immunofluorescence demonstrated that 29.54% of the SKPs expressed MAP-2 and no SKPs expressed NeuN.Conclusion：The reprogrammed neural stem cells can be prepared through transfecting neurogenin2 into SKPs via plentivirus in vitro,and their capability of differentiating into neural cells elevated visibly after neurogenin 2 transfection.