Calcitonin Gene-related Peptide Stimulates Neuronal Differentiation Of Neural Stem Cells In Vitro

Objiective: To investigate the effects of calcitonin gene-related peptide(CGRP) on the proliferation and neuronal differentiation of neural stem cells(NSCs) isolated from the newborn mice in vitro.Methods: Neural stem cells were isolated enzymatically and mechanically from Subventricular zone(SVZ) of neonatal mice and cultured as free-floating cell aggregates named neurospheres in the presence of epidermal growth factor(EGF) and basic fibroblastic growth factor(b FGF). Immunocytochemical stainings of anti-Nestin and anti-SOX2 were used to identify NSCs. To find the appropriate time when NSPCs proliferated quickly, MTT assay was used. In order to investigate the effects of CGRP on the proliferation of NSPCs, the Brd U pulse-label method and the method of quantification of the average volume of neurospheres were used. For the role of CGRP on neuronal differentiation of neural stem cells, the proportions of Tuj-1-positive cells were counted and compared among the four groups.Results: The results of MTT revealed that NSCs proliferated more quickly after cultured for 4 days comparing to other cultured time in vitro. After cultured with growth factors for four days, the number of proliferated NSCs in 100 n M CGRP group was to equal to other 3 groups. Quantification of the average volume of neurospheres in four groups uncovered that neurospheres in 100 n M CGRP group were equal large to other three groups. Three days post cultured in vitro, the percentage of Tuj-1-positive neurons in control group, 1n M CGRP group, 10 n M CGRP group and 100 n M CGRP group were determined to be(26.1±5.1) %,(28.1±6.1) %,(34.7±4.8) % and(56.0±5) %, respectively. The percentage of Tuj-1-positive neurons in 100 n M CGRP group was significantly higher than other three groups(P<0.05). However, after cultured for 7 days in vitro, the percentage of Tuj-1-positive neurons in control group, 1n M CGRP group, 10 n M CGRP group and 100 n M CGRP group were determined to be(45.4±4.2) %,(53.8±4.3) %,(70±2.6) % and(48.8±4.1) %, respectively. The percentage of Tuj-1-positive neurons in 10 n M CGRP group was significantly higher than other three groups(P<0.05).Conclusion: The neural stem cells from newborn mice SVZ are successfully isolated, cultivated and identified. While CGRP can not promote the proliferation of NSCs, CGRP stimulates neuronal differentiation of NSCs in a dose- and time-depend manner in vitro.