关键词: 重组人粒细胞集落刺激因子, 神经干细胞, 信号转导与转录激活因子3, 免疫荧光, 免疫印迹法, 小鼠

Abstract: Objective To investigate whether granulocyte colony-stimulating factor (G-CSF) has a direct effect on the proliferation of mouse neural stem cells EM>in vitro/EM>. Methods Primary mouse neural stem cells(NSCs) were isolated from Kunming mice and cultured in serum free medium. The third passage NSCs were used in the experiments. NSCs were treated with G-CSF (10, 30, 60, 100, and 200μg/L). The proliferation of NSCs was detected by MTT colorimetric assay and bromodeoxyuridine (BrdU) incorporation assay. The expressions of STAT3 and p-STAT3 were examined by Western blotting. Results There was the expression of G-CSF receptor on the neural stem cells. G-CSF obviously improved the viabilities of NSCs in a dose-dependent manner. Furthermore, 100μg/L G-CSF yielded the optimal effect. The BrdU-positive cells against total NSCs treated with G-CSF were markedly enhanced. Time course experiments demonstrated that STAT3 phosphorylation by G-CSF was evident after 5 minutes, reaching the maximum after 30 minutes. At 75 minutes, the effect on STAT3 returned to the basal value. Pretreatment with the G-CSF receptor antibody significantly reduced the phosphorylation of STAT3 induced by G-CSF. BrdU incorporation showed that cell proliferation in the presence of G-CSF and neutralizing antibody for G-CSF receptor was similar to that of the reduced growth medium group. Conclusion G-CSF may stimulate the proliferation of NSCs EM>in vitro/EM>. Our results provide the cellular basis of the role of G-CSF in NSCs EM>in vitro/EM>, which may serve as a useful reference for future studies of the powerful neuroprotective effect of

Key words: Recombinant human granulocyte clony simulating factor, Neural stem cell, Signal treansducer and activator of transcription 3, Immunofluorescence, Western blotting, Mouse