| Objective: To explore the effects of combination of bFGF, PDGF, Ra, HRG, and Forskolin under the condition of serum-free on inducing the differentiation of NSCs.Methods: The NSCs of rats were separated from the brains of rat embryos of 14-16 days old and cultured by using the serum-free technology. NSCs were certified by immunocytochemistry, and the proliferation cycles of them were detected by flowcytometer. After two or three passages, NSCs spheres were plated on PLL-coated coverslips in 24 well dishes, and were divided into 4 groups (two steps group, one step group, serum group, and bFGF group) cultured in different media. Map2 immunofluorescence staining was used to detect the differentia-tion of NSCs into neurons. The perimeters and areas of cell bodies', the lengths of longest process's and the induced differentiation rate of Map2 positive cells were obtained by using image analysis system. Whole cell patch-clamp technique was performed to record the current of sodium ion of neuron-like cells'. The software of STATA 7.0 was applied to do variance and comparisons of every two groups. Results: 1. The dissociation, culture, observation and identification of NSCs: The cells dissociated from cerebral cortex of rat embryos of 14-16 days old were round and bright. They could be passaged after being cultured six to seven days. The edges of the NSCs spheres were sharp. The immunocyto-chemistry results showed that neurospheres were nestin-positive. Detection of flowcytometer indicated that many cells were at the period of G1/M and S.2.The differentiation of NSCs into neurons: The immunocytochemistry results showed the proportion of Map2 positive cells' in one step group, being 43.35±13.75, was higher than that of other groups, and there were many TH-positive cells in this group. The cells bodies were big in one step and two steps group, and the processes were long and abundant, the areas of them were 136.22±33.08μm~2 and 131.45±24.58μm~2,bodies' perimeters were 48.63±5.5μm and 49.3±7.71μm, the lengths of the longest process of each neuron-liked cells were 123.63±31.48μm and 116.51±38.34μm. These statistics results manifested that there were significant differences on percentage of Map2 positive cells between one step and serum group (P＜0.05), but there weren't significant differences among other group; that there were significant differences on the lengths of the longest process of the Map2 positive cells between one step, serum, and bFGF group but there weren't differences between one step and two steps group; that there weren't significant differences on the cell bodies' perimeters and areas of the Map2 positive cells' between one step and two steps group also between serum and bFGF group, but there were significant differences among other groups. The electrophysiology results showed that most of the morphological maturity neuron-like cells (80%)could be stimulated to show the current of sodium ion in one step group and a less cells (20%) had this property in two steps group, The cells of other groups hadn't this property.Conclusion: 1.The cells from cerebral cortex of rat embryos of 14-16 days possessed the ability of self-renew and multi-potential. They belonged to neural stem cells. 2. It was feasible to induce NSCs to neurons under the condition of serum-free combinating application of bFGF, PDGF, Ra, HRG, and Forskolin. 3.Under the condition of serumfree, the neuron-like cells differentiated from one step group was more mature and the differentiation rate of neuron in one step group was bigger as compared with two step group.
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