[The effects of optical genetic techniques on new neurons through the Wnt/ß-Catenin pathway].

ABSTRACT

OBJECTIVE: To investigate the effects of optical genetic techniques on new neurons through the Wnt/ß-Catenin pathway. METHODS: Neural stem cells (ESCs)were extracted from the cerebral cortex of fetal rat and transfected by lentivirus carrying DCX-ChR2-EGFP gene and the expression of DCX of newborn neurons differentiated from neural stem cells were observed. All cells were divided into 3 groups(n=9) control group, NSCs+EGFP and NSCs+ChR2 groups. The control group was normal cultured NSCs (NSCs group); the neural stem cells in NSCs+EGFP group were transfected with lentivirus carrying EGFP gene. The neural stem cells in NSCs+ChR2 group were infected with lentivirus carrying DCX-ChR2-EGFP gene. After 48 hours of lentivirus infection, 470 nm blue laser irradiation was performed for 3 consecutive days. NeuN+ positive cell density(the maturation of neural stem cells)and the ratio of NeuN+/Hoechst in each group were observed. Western blot was used to detect the expression levels of MAP2, NeuN, Neurog2, NeuroD1 and GluR2. Western blot was used to detect the expressions of ß-catenin and TCF4 associated with Wnt/ß-catenin signaling channel. Verapamil (100 µmol/L, L-type calcium channel blockers) and Dkk1 (50 µg/ml, ß-catenin inhibitor) were used to treat stem cells of the NSCs+ChR2 group and then the expressions of MAP2, NeuN, Neurog2, NeuroD1 and GluR were detected by Western blot. RESULTS: After 3 days of 470 nm blue laser irradiation, NeuN+ positive cell density(the maturation of neural stem cells)and the ratio of NeuN+/Hoechst, the expression levels of the protein MAP2, NeuN, Neurog2, NeuroD1, GluR and the protein ß-catenin and TCF4 associated with Wnt/ß-catenin signaling channel detected by Western blot were significantly increased in the group of NSCs+ChR2, compared with NSCs and NSCs+EGFP groups. The expressions of MAP2, NeuN, Neurog2, NeuroD1 and GluR were remarkably decreased after treated by verapamil and Dkk1 in the group of NSCs+ChR2. It was proved that the opening of ChR2 channel producing cationic influx promoted the maturation of neural stem cells and induced by the Wnt/ß-catenin signaling pathway. CONCLUSION: Optical genetic promoted the maturation of newborn neurons through the Wnt/ß-catenin signaling pathway.