大鼠侧脑室下区神经干细胞具有不易兴奋性

目的建立体外培养大鼠侧脑室下区神经干细胞的方法，观察大鼠侧脑室下区神经干细胞的膜兴奋性。方法无血清培养方法体外分离、纯化孕15～16dwistar胎鼠的侧脑室下区神经干细胞，用免疫荧光鉴定干细胞标记蛋白nestin表达情况、用tuj-1和GFAP免疫染色研究体外NSC的分化情况；取第二代神经干细胞给予DiBACA（3）染色后，经高浓度氯化钾刺激，激光共聚焦显微镜动态扫描，观察侧脑室神经干细胞的兴奋性。结果采用无血清培养基体外分离的神经干细胞具有自我增殖、多向分化潜能等干细胞一般特点，且表达干细胞的标记蛋白nestin；采用DiBAC4（3）染色，高浓度钾刺激后，细胞荧光强度无显著变化，即细胞膜电位无明显改变，神经干细胞具有不易兴奋性。结论采用无血清培养方法成功分离扩增大鼠脑内神经干细胞；由大鼠侧脑室分离而来的神经干细胞具有不易兴奋性。

The Excitability Characteristic of Neural Stem Cells in the Subventricular Zone of Rats

Objective To establish the methods of culturing neural stem cells isolated from ＇subventricular zone of rats in vitro and investigate the excitability Of neural stem cells. Methods Cells from El6 wistar rat subventricular zone were cultured in proliferative medium containing EGF and bFGF and the neural stem cell were separated and purified with passages. Cells were cultured within 4 hours on coverslips coated with poly-L-lysine with DMEM/F12 medium containing EGF and bFGF before the experiment. The membrane potential changes of NSCs were detected with fluorescent dye DiBACA（3 ） （bis-（ 1,3-dibutylbarbituric acid） trimethine oxonol） by confocal laser scanning microscope when stimulated by KC1. Results Neural stem cells formed neurosphere in specific medium and presented nestin-positive by immunostaining, and it survived for 3-4 passages. When the medium was added with fetal calf serum, the neural stem cells can be differentiated into neuron （ immunostained with tuj-1 ） and astrocytes （ immunostalned with GFAP）. Membrane potential reflected indirectedly by the changes of fluorescent intensity of DiBAC4 （3） stain, after KC1 stimulation changed slightly. Conclusions The neural stem ceils were obtained successfully by using proliferative medium containing EGF and bFGF in virto. The membrane potential of neural stem cell was inexcitable.