再程序化星形胶质细胞的制备及体外定向分化为神经元的研究

Research on preparation and neuronal induction of reprogrammed astrocytes in vitro

目的探讨再程序化星形胶质细胞制备并在体外诱导其分化为神经元。方法在体外培养大鼠脑皮质来源星形胶质细胞(astrocyte),随后将提纯、鉴定过的第三代星形胶质细胞接种于12孔培养皿中,并分为A、B、C 3组。其中A组为带有绿色荧光蛋白(GFP)的慢病毒载体介导neurogenin2(Ngn2)基因转染的星形胶质细胞,制备再程序化星形胶质细胞;B组为带有GFP基因的空载体病毒转染的星形胶质细胞;C组为未进行慢病毒介导基因转染的星形胶质细胞;转基因1周后加入含细胞生长因子诱导培养基诱导分化15 d,光镜下观察各组细胞形态变化以及定向神经元分化的差异。结果 A组星形胶质细胞转基因后再诱导15 d,很大部分细胞形态呈神经元样改变,胞体呈梭形或椭圆形,有多个突起伸出且突起较长,表达神经元核蛋白(Neu N)、神经丝蛋白(NF)及神经元特异性烯醇化酶(NSE)的比例大大提高,相比B组及C组,差异有统计学意义(均P<0.05);而B组与C组神经元分化比例的差异无统计学意义(P>0.05)。结论慢病毒介导Ngn2基因体外转染星形胶质细胞可制备出再程序化星形胶质细胞,诱导后具有更强的向神经元定向分化能力。

Objective To study the preparation and neuronal induction of reprogrammed astrocytes in vitro. Methods Separated from rat cerebral cortex,astrocytes were purified,identified,three passaged,seeded in 24-well culture plates and divided into three groups in terms of tranfection.Group A were transfected with neurogenin2（ Ngn2） via green fluorescence protein（ GFP）-plentivirus,thus reprogrammed ASTs were prepared. Group B were transfected only GFP-plentivirus. Group C were cultured in nontransfected condition. All of the groups were induced for 15 days in the medium supplemented cell growth factors following 7 days ＇transfection. The induced cells morphous was observed by microscope,and the marker of neuronal neuronal special neuclear protein（ Neu N）,neuron specific enolase（ NSE）,neurofiliament（ NF） were detected by immunofluorescense. Results After transfection,the shape of ASTs got fusiform or eclipse after fifteen days of inducement,and took on neuron-like. Every cells had long bipolar or tripolar apophysises. Compared to both Group B and Group C,the percentage of Neu N,NSE positive cells were pronounced elevated（ all P〈0. 05）,while the efficiency in Group B and Group C was not significant,P〉0. 05. Conclusion The programmed ASTs are prepared through transfecting Neurogenin2 via plentivirus in vitro,and their potential of differentiation into neuron is considerably enhanced.