神经干细胞向神经元诱导和分化的研究

为探讨无血清条件下联合应用bFGF、PDGF、RA、HRG、Forskolin对大鼠神经干细胞诱导分化的作用,本研究采用体外分离、培养神经干细胞,传2~3代后,联合应用因子体外诱导神经干细胞分化等方法;通过Ⅱ型微管相关蛋白(MAP2)和S100抗体免疫荧光染色鉴定分化的细胞;图像分析系统测量神经元样细胞的参数;全细胞膜片钳技术记录分化后细胞的钠电流。结果显示:MAP2阳性神经元样细胞所占比例,联合因子组明显高于血清组(P<0.05);胞体面积及周长、突起的长度,联合因子组明显高于血清组和bFGF组(P<0.05)。另外,联合因子组中80%形态似神经元样的细胞能诱发电压依赖性的Na+通道电流。以上结果表明在体外无血清条件下联合应用bFGF、PDGF、RA、HRG、Forskolin对大鼠神经干细胞向神经元定向诱导分化是可行的,分化的神经元比例高,且细胞更具有神经电生理特性。

STUDY OF THE INDUCTION AND DIFFERENTIATION OF RAT NEURAL STEM CELLS INTO NEURONS

In order to explore the effects of combination of bFGF, PDGF, RA, HRG, Forskolin under the condition of serum-free on inducing the differentiation of NSCs from the brains of rat, the NSCs were separated and cultured in vitro, after two or three passages it were induced in united factors media. The microtubule associate protein Ⅱ (MAP2) and S100 immunofluorescence staining was used to detect the differentiated cells. The cell parameters of MAP2 positive neuron-like cells were obtained by using image analysis system. Whole cell patch-clamp technique was performed to record the current of Na~+. The present results showed that the percentage of MAP2 positive cells was apparently increased in comparison with serum group (P<0.05); the cell bodies’perimeter, area and the length of the longest process of the MAP2 positive cells were obviously higher in comparison with serum and bFGF group(P<0.05). On the other hand, most of the neuron-like cells (80%) can be stimulated to show the current of sodium ion in united factors group. The above results indicate that it was feasible to induce NSCs to neurons under the condition of serum-free combinating application of bFGF, PDGF, RA, HRG and Forskolin, the proportion of differentiated neuron was higher and the differentiated neurons have more electrophysiological characteristics.