Objective: To explore the expression of Wnt-1 during the process of inducing neural stem cells (NSCs) into neurons by using all-trans-retinoic acid (ATRA) in vitro and the effect of Wnt-1 on NSCs differentiation.
Methods: NSCs isolated from cerebral cortex of SD rat embryo (12-16 days' gestation) were cultured. The concentration of cells at passage 3 were adjusted to 1 x 10(6) cells /mL and treated with ATRA at 0.5, 1.0, 5.0 and 10.0 micromol/L, respectively. Differentiation ratio of NSCs into neurons in each group was detected by double-labelling immunofluorescence technique and flow cytometry, and 1.0 micromol/L was selected as the best concentration for ATRA to promote NSCs differentiation. In experimental group, NSCs at passage 3 were cultured with ATRA at 1.0 micromol/L in vitro, and expression of Wnt-1 was detected by immunocytochemistry staining, real-time fluorescent quantitive PCR and Western blot at 3, 5, 7 and 9 days after culture, respectively. The cells at passage 3 receiving no ATRA served as control group.
Results: Immunocytochemistry staining: in the control group, there was little Wnt-1 protein expression; in the experimental group, peak expression of Wnt-1 and numerous positive cells occurred at 3 days after culture, the positive expression of Wnt-1 was still evident at 5 days after culture, and there was significant difference between two groups in integrated absorbance (IA) value at 3 and 5 days after culture(P < 0.05), obvious decrease of positive expression of Wnt-1 was evident, and no significant difference was evident between two groups in IA value at 7 and 9 days (P > 0.05). Real-time fluorescence quantitative PCR: the relative expression of Wnt-1 mRNA in the control group was 0.021 7 +/- 0.072 1; the relative expression of Wnt-1 mRNA in the experimental group at 3, 5, 7 and 9 days was 0.512 2 +/- 0.280 0, 0.216 4 +/- 0.887 0, 0.038 5 +/- 0.299 4 and 0.035 5 +/- 0.309 5, respectively, indicating the value decreased over time, and there were significant difference between two groups at 3 and 5 days (P < 0.05), and no significant difference at 7 and 9 days (P > 0.05) . Western blot detection: specific and visible staining band was noted; in the control group, Wnt-1 protein expression was 0.005 1 +/- 0.558 3; in the experimental group, Wnt-1 protein expression at 3, 5, 7 and 9 days was 0.451 7 +/- 0.071 3, 0.311 7 +/- 0.080 5, 0.007 3 +/- 0.052 7 and 0.004 7 +/- 0.9314, respectively, suggesting the value decreased over time; there were significant differences between two groups at 3 and 5 days (P < 0.05), and no significant differences at 7 and 9 days (P > 0.05).
Conclusion: With the induction of ATRA at 1.0 mmicromol/L, Wnt-1 and NSCs differentiation in early stage are positively correlated. Its possible mechanism may rely on the activation of such signals as classic Wnt-1 signal pathway, indicating Wnt-1 relates to the differentiation of NSCs into neurons.