摘要
目的制备再程序化脂肪源性干细胞以及探索诱导其定向分化为神经元的方法。方法体外培养脂肪来源干细胞(ADSCs),取纯化、鉴定过的第3代ADSCs接种于24孔板中并分A,B,C三组:A组为带有绿色荧光基因(GFP)的慢病毒载体介导Neurogenin2(Ngn2)基因转染的ADSCs,制备再程序化脂肪干细胞;B组为带有GFP的空载体病毒转染的ADSCs;C组为未进行慢病毒介导基因转染的ADSCs;转基因7d后加入含细胞生长因子诱导培养基诱导分化15d。光镜下观察各组细胞形态变化以及免疫荧光研究各组ADSCs诱导后定向分化为神经元的差异。结果与B组和C组相比,A组ADSCs转基因后再经诱导分化15d后绝大部分细胞类似神经元,胞体呈梭形或椭圆形,有两个或三个突起伸出,细胞表达神经丝蛋白NF、神经元特异性蛋白NeuN及神经元特异性烯醇化酯酶NSE比例大大提高。B组与C组的神经元分化效率,无显著差异,P>0.05。结论慢病毒介导Neurogenin2基因体外转染ADSCs可以制备出再程序化脂肪源性干细胞,诱导后具有更强的定向分化为神经元的能力。
Objective To discuss the preparation and neuronal induction of reprogrammed adipose derived stem cells in vitro. Methods Isolated from rat inguinal adipose tissues , adipose- derived stem cells （ADSCs） were purified, identified three passaged, seeded in 24-well culture plates and divided into three groups in term of transfection. Group A were transfeeted with Neurogenin2 via GFP-plentivirus,thus reprogrammed ADSCs were prepared, group B were transfected with only GFP- plentivirus;group C were cultured in nontransfected condition. All of the groups were induced for fifteen days in culture medium supplemented cell growth factors following seven clays of transfection in common medium. The induced cells morphous was observed by microscope, and the marker of neuronal NeuN, NSE, NF were detected by immunofluorescence. Results After transfection, the shapes of ADSCs got fusiform or eclipse after fifteen days of inducement, and took on neuron-like. Every cells had bipolar or tripolar apophysises. Compared to both group B and group C, the percentage of the NF, NeuN and NSE positive cells was evidently raised, while the efficiency in both group B and group C is not different, P 〉 0. 05. Conclusion The reprogrammed ADSCs are prepared through transfeeting neurogenin2 via plentivirus in vitro, and their potential of differentiation into neuron is considerably enhanced.
出处
《临床神经外科杂志》
CAS
2012年第3期129-132,共4页
Journal of Clinical Neurosurgery
基金
国家自然科学基金面上项目(30973092
81171147)
江苏省"兴卫工程"医学重点人才基金[20072011]
江苏省"六大人才高峰"基金2010
关键词
脂肪源性干细胞
神经元分化
再程序化
adipose derived stem cells
neuronal differentiation
reprogammed
