rhG-CSF促进体外培养神经干细胞的增殖

rhG-CSF promoting the proliferation of neural stem cells in vitro

目的探讨重组人粒细胞集落刺激因子(rhG-CSF)对小鼠胚胎神经干细胞(NSCs)增殖的作用。方法分离培养小鼠NSCs,通过向培养基中添加G-CSF(10、30、60、100和200μg/L),四甲基偶氮唑蓝(MTT)比色法以及5-溴-2'-脱氧尿苷(BrdU)免疫荧光染色标记检测NSCs的增殖。免疫印迹法检测NSCs增殖时信号转导与转录激活因子3(STAT3)和磷酸化信号转导和转录激活因子3(p-STAT3)的表达。结果神经干细胞表达G-CSF受体,细胞活性随G-CSF的浓度不同表现出一定的剂量依赖性,当G-CSF的浓度为100μg/L时细胞的活性最高。G-CSF处理组的细胞增殖活性明显高于对照组。进一步证实,在G-CSF处理后,p-STAT3的表达则在5 min开始升高,30 min到达峰值,之后开始下降,至75 min接近正常水平。加入G-CSF受体的抗体后,p-STAT3的活化现象消失,并且细胞的增殖也明显低于G-CSF作用组,和对照组接近。结论 rhG-CSF可促进小鼠NSCs的增殖,其作用机制可能是通过细胞表面的G-CSF-R促进STAT3的活化。G-CSF可能作用于内源性NSCs的活化和增殖。

Objective To investigate whether granulocyte colony-stimulating factor （G-CSF） has a direct effect on the proliferation of mouse neural stem cells in vitro. 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 ceils 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 in vitro. Our results provide the cellular basis of the role of G-CSF in NSCs in vitro, which may serve as a useful reference for future studies of the powerful neuroprotective effect of G-CSF in various types of neurological disorders.