The effect of R-spondin3 on proliferation of neural stem cells and Wnt/β-catenin signals in mice

Objective To explore the effect of R-spondin3 on the proliferation of neural stem cells (NSCs) and its mechanism in mice. Methods The mouse NSCs derived from the subventricular zone of E14-15d CD1 mice were confirmed by immunofluorescence assay. The NSCs after 3 passages of culture were chosen and randomly divided into 2 groups (V=1 mL). In the experimental group, 0.8 μL of R-spondin3 with an initial concentration of 50 μg/mL was added (final concentration: 40 ng/mL) while in the control group an equal amount of culture fluid was added. The proliferation of the cells in the 2 groups was detected by 5-Bromo-2-deoxy Uridine (BrdU) kits after the cells were treated by R-spondin3 for 6 hours. The protein expression of β-catenin was measured by western blotting after the cells were treated by R-spondin3 for 4 and 8 hours. Results Under optical microscopy, the round and bright cells grew in culture medium and easily accumulated to become neurospheres. Immunofluorescence assay showed that over 90% of the cells expressed Nestin and SOX2 and that some of them expressed NeuN or GFAP after induced differentiation. Brdu proliferation test showed that the proliferation rate of Brdu+/DAPI+ for the experimental group (1.56±0.03) was significantly higher than that for the control group (1.04±0.04) (P<0.05). Western blotting showed that the expression levels of β-catenin were increased at both 4h and 8h after treatment for the experimental group (1.09±0.10 and 1.20±0.13), significantly higher than those for the control group (0.56 ± 0.05 and 0.83 ± 0.04) (P<0.05). Conclusions R-spondin3 can promote in vitro proliferation of NSCs in mice, which may be associated with activated Wnt/β-catenin signal pathways. Key words: R-spondin3; Neural stem cells; Wnt/β-catenin signal pathway