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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 study the feasibility of preparing skin-derived reprogrammed neural stem cells and induce them to differentiate into nerve cells. Methods: Skin-derived precursors (SKPs) from rat skin were cultured and purified in vitro. Lentiviral vector system containing green fluorescent protein (GFP) gene was used as a gene transfection tool. SKPs Group 3: SKPs transfected with lentivirus-mediated neurogenin2 (Ngn2) gene in group A; SKPs transfected with empty vector lentivirus in group B; SKCs transfected with lentivirus-mediated gene in group C; Induction medium was added for 7 days with addition of 10% fetal bovine serum without growth factor. The morphological changes of cells in each group were observed under light microscope and the differences of directional differentiation into nerve cells after SKPs induction in each group were studied by immunofluorescence microscopy. Results: The green fluorescence reached the peak on the 7th day after transfection of SKPs in Group A, and the green fluorescence was consistently and stably expressed to the 8th generation. After 7 days of induction, the majority of cells were similar to neurons. The cell bodies were fusiform or oval with multiple The results of immunofluorescence showed that 90.98% of cells expressed microtubule-associated protein-2 (MAP-2) and 62.23% of cells expressed neuron-specific protein NeuN. The SKPs in group B showed green fluorescence after 7 days of transfection of empty virus. Most of the cells showed no significant changes after 7 days of induction compared with that before induction. The results of immunofluorescence showed that 34.35% of the cells expressed MAP-2 and no neuron-specific protein NeuN expression. There was no green fluorescence on SKPs in Group C; Most of the cells showed no significant changes after 7 days of induction compared with that before induction. Immunofluorescence showed that 29.54% of cells expressed MAP-2 and no Neuron-specific protein NeuN. CONCLUSION: Lentivirus-mediated Ngn2 gene transfection into SKPs can prepare skin-derived reprogrammed neural stem cells and enhance their ability to differentiate into neurons.