Abstract: Objective To investigate the effect of LINGO-1 shRNA (LV) and neurotrophic factors cocktail (NTFs,NTFcocktail) encapsulated by Pluronic F-127(PF-127)hydrogel (PF-127-LV-NTFs)three-dimensional(3D) composite scaffolds on the proliferation and differentiation of rat neural stem cells. Methods The LV was constructed and infected with NSCs at different MOI. RT-qPCR and Western blot were used to detect the expression of LINGO-1 in NSCs. Brain derived neurotropic factor(BDNF), neurotrophin-3(NT-3), platelet-derived growth factor(PDGF),insulin-like growth factor 1(IGF-1), epidermat growth factor(EGF), basic fibroblast growth factor(bFGF) and glial cell derived neurotrophic factor(GDNF) were prepared as NTFs. The LV and NTFs were mixed with PF-127 and divided into different groups to culture NSCs. CCK-8 was used to detect the cell viability of NSCs, the survival rate of NSCs was detected by LIVE/DEAD staining, and immunofluorescence staining was used to detect the proliferation and differentiation of NSCs. Results The optimal MOI for LV was 5. CCK-8 assay results showed that there was no significant difference in the survival rate of NSCs. The LIVE/DEAD and Nestin immune-fluorescence staining results showed that compared with control, the survival rate of NSCs and the proportion of neuron differentiation positive were the highest in PF-127+LV+NTF group(P＜0.01). Conclusions PF-127-LV-NTFs 3D composite scaffold is favouring to support the proliferation and the directional differentiation of NSCs of rat.

Key words: neural stem cells, LINGO-1 shRNA, PF-127 hydrogel, neuronal differentiation