神经前体细胞的分离培养及绿色荧光蛋白基因的转染

Isolation and Culture of Neural Precursor Cells and Transfection of Green Fluorescence Protein Gene

【目的】为替换中枢神经损伤后死亡的神经元提供方便追踪观察的神经前体细胞。【方法】应用含碱性成纤维生长因子 (bFGF)的无血清培养技术 ,从新生大鼠海马组织分离培养神经前体细胞 ,并借助免疫组织化学技术检测这些细胞巢蛋白 (nestin)的表达以及细胞分化后神经丝蛋白 (NF)和胶质原纤维酸性蛋白 (GFAP)的表达。采用磷酸钙法将绿色荧光蛋白(GFP)基因转染目的细胞内。【结果】从海马组织分离的细胞具有分裂能力 ,能表达nestin。分化后一些细胞能表达NF ,而另一些细胞则表达GFAP。GFP基因修饰的细胞能发出荧光。【结论】从海马组织分离的细胞能表达nestin ,属于神经前体细胞 ,它们具有明显的增殖能力。有些神经前体细胞已经分化为神经元和神经胶质细胞。GFP基因已成功转染到神经前体细胞内 ,并表达了GFP。

To provide easily tracing and observing neural precursor cells for replacing dead neurons after central nervous system injured. Neural precursor cells from hippocampic tissue of neonate rat were isolated and cultured by serum free medium containing basic fibroblastgrowth factor (bFGF). The expression of nestin of the cells and the expression of neurofilament (NF) or glial fibrillary acidic protein (GFAP) of differentiated cells were detected by immunohistochemistry. The gene of green fluorescence protein (GFP) was transfected to the target cells with calcium phosphate transfection. The cells from hippocampic tissue had the potential of proliferation and expressed nestin. Some differentiated cells showed NF positive, others were GFAP positive. The cells modified by GFP gene showed fluorescence. [Conclusion] The cells from hippocampic tissue of neonate rat cultured in vitro belong to the neural precursor cells which can express nestin and have the potential of proliferation. Some neural precursor cells had differentiated into neurons and neuroglial cells. The GFP gene has been successfully transfected to the neural precursor cells and effectively expressed.