Abstract:

Objective To investigate the influence of 17β-estradiol （17β-E2） in the proliferation and differentiation capabilities of primary cultured hippocampal neural stem cells （NSCs），and to clarify its potential mechanism. Methods The NSCs were isolated from the hippocampal tissue of SD rats within 24 h of birth，and divided into control group and 17β-E2 group. Immunofluorescence method was used to detect the expressions of Nestin and 5-ethynyl-2'-deoxyuridine （EdU） in NSCs in two groups， and the proliferation rates of NSCs were calculated. Western blotting method was used to detect the expression level of Nestin protein. Flow cytometry was used to analyze the percentages of NSCs at different cell cycles. Immunofluorescence method was used to identify the expressions of markers for neurons βⅢ-tubulin and astrocyte marker glial fibrillary acidic protein （GFAP） in the NSCs after differentiation， and the relative ratio of neurons to astrocytes was calculated. Western blotting method was used to detect the expression levels of βⅢ-tubulin and GFAP proteins as well as phosphatidylinositol 3-kinase （PI3K）， protein kinase B （Akt）， phosphorylated Akt （p-Akt）， glycogen synthase kinase-3 beta （GSK-3β）， phosphorylated GSK-3β （p-GSK-3β）， and beta-catenin （β-catenin） proteins related to PI3K/Akt/GSK-3β pathway. Results Immunofluorescence assay revealed positive Nestin expression in NSCs in two groups； compared with control group，the proliferation rate of NSCs in 17β-E2 group was increased （P<0.01）. Compared with control group， the expression level of Nestin protein in the NSCs in 17β-E2 group was increased （P<0.05），and the percentage of NSCs in S phase was increased （P<0.01）. Compared with control group，the relative ratio of neurons to astrocytes in 17β-E2 group was significantly increased （P<0.05）. After differentiation，compared with control group，the expression level of βⅢ-tubulin protein in the NSCs in 17β-E2 group was increased （P<0.05）， and the expression level of GFAP protein was decreased （P<0.01）， while the expression levels of Akt， p-Akt， β-catenin， and p-GSK-3β proteins were increased （P<0.05 or P<0.01）， and the expression level of GSK-3β protein was decreased （P<0.05）. Conclusion 17β-E2 can promote the proliferation of NSCs and facilitate their differentiation towards neurons，and its mechanism may be related to the PI3K/Akt/GSK-3β signaling pathway.

Key words: 17β-estradiol, Neural stem cells, Cell proliferation, Cell differentiation, Phosphatidylinositol 3-kinase, Protein kinase B, Glycogen synthase kinase-3 beta