| Granulocyte-colony stimulating factor (G-CSF), a member of the growth factor family, mainly stimulates the differentiation of committed progenitor cells to neutrophils and also modulates neutrophil actions and their distribution in the body. It has been used clinically to facilitate hematopoietic recovery after bone marrow transplantation, to mobilize peripheral blood progenitor cells in healthy donors, and to treat severe congenital neutropenia. Available data suggest that there is little doubt that G-CSF is a safe agent for use in the human population. Many patients have received G-CSF over the last decades after myelosuppressive treatment. It also has trophic effects on the different cell types, including neuros. Recent experimental studies have shown that the administration of G-CSF is neuroprotective, while the mechanism is still unclear.G-CSF and its receptor are widely expressed in the adult central nervous system (CNS) in rat. It has been revealed that permanent middle cerebral artery occlusion resulted in increasing levels of G-CSF mRNA compared with the normal cortex. Recently, G-CSFR is reported expressed on radial glia cells throughout cortical neurogenesis, which suggests G-CSF may play key role in the development of CNS. The effect of G-CSF on neural stem cells (NSCs) and the possible mechanism is remained to be ascertained.Expanding evidence from recent studies shows that G-CSF exerts neuroprotective effects including mobilization of hematopoietic stem cells to the injured brain and antiapoptotic effect. A question that may be raised by these observations is whether the endogenic NSCs are mobilized by G-CSF to promote the recovery of brain injury. The present study is aimed to investigate the effects of G-CSF on the NSCs in vitro. For the aim, immunofluorescence, Western blot and RT-PCR were used to analyse the effect and the mechanism of G-CSF on NSCs. Further we established the model of hypoxia-ischemia brain damage and investigated the effect of G-CSF on endogenic NSCs, which may show G-CSF to be a novel therapeutic drug for future using on brain injury.1. The effects of G-CSF on the neural stem cells in vitroNeuroepithelial cells from neural tube are the most primitive NSCs. These neural stem cells (NSC) are a population of undifferentiated cells that are capable of extended self-renewal and multipotential ability to generate multilineage cell types. At E12.5d mice, NSCs suspension was successfully isolated by stereoscopic microscope and mechanical blowing, and neural progenitor cells gained in serum-free medium. BrdU labeling technique and nestin immunofluorescence staining technique were used to detect self-duplication and self-renewal of the cells. Microtubule associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) immunofluorescence staining technique were used to identify differentiation. The results suggest that cells from brain cortex of murine embryos are NSCs and are embryonic mice are easily extracted and naturally differentiate into neurocyte and neuroglial cells. The primary NSCs were used to detect the expression of G-CSF receptor (G-CSFR). To investigate the effects of G-CSF, the cells were treated with G-CSF with the concentration of 10,30, 60 100 and 200 ng/ml respectively in cultures for 24 and 48 h and cell viability was determined by MTT assay. The result showed that G-CSF obviously improved the viabilities of NSCs in a partly dose-dependent manner and the optimal concentration of G-CSF 100 ng/ml yielded. The BruU-positive cells against total NSCs treated with G-CSF was markedly enhanced, suggested that G-CSF stimulates the proliferation of NSCs. We next determined the effect of G-CSF on the differentiation on NSCs. It showed that the expression level of MAP2 and GFAP in G-CSF treated group at day 3 and day 5 of culture were significantly higher than that in the corresponding untreated control group.2. The possible mechanism of G-CSF promoting the proliferation and differentiation on NSCsWe examined the STAT signaling activation in NSCs proliferation induced by G-CSF. Time course experiments demonstrated that STAT3 phosphorylation induced by G-CSF was evident after 5 min, reaching the maximum after 30 min. At 75 min, the effect on STAT3 returned to the basal values. To further ascertain the functional role of STAT3 in NSCs activated by G-CSF, the latter was neutralized with its receptor antibody. It was found that pretreatment with G-CSF receptor antibody significantly reduced the phosphorylation of STAT3 induced by G-CSF. BrdU incorporation experiment showed that cell proliferation in the presence of G-CSF and neutralizing antibody for G-CSF receptor was similar to that in the reduced growth medium group.To determine the mechanisms of G-CSF promoting the differentiation of the NSCs, the expression of bHLH transcription factors that might be correlated with neurogenesis and astrocytogenesis was detected. The mRNA levels of Neurogl, NeuroD2, Mash1, Id1 and Hes5 were significantly increased in G-CSF-treated groups than those in the untreated control groups and the peak points were earlier and higher than those in control groups. We found interestingly that G-CSF did not alter NSCs fate in an undifferentiated state.3. The neuroprotective effect of G-CSF on hypoxia-ischemia brain damage modelSome studies provide evidence that G-CSF exerts an antiapoptotic effect on neurons, while the mechanism was still unclear. At present, there are no effective means of repairing the hypoxic-ischemic brain damage (HIBD) once damage has occurred. We established neonatal mice model with severe HIBD. The neonatal KunMing mice aged 7 days, after anesthetized their left common carotid arteries were double-ligatured and cut between the ligatures, were exposed to 120 minutes of hypoxia (8%O2+92%N2) by being placed in a chamber partially submerged in 37℃. The water content of left hemisphere after HIBD increased significantly compared with the right hemisphere. The water content in G-CSF group obviously reduced compared with the HIBD group at the same time point. After 24 hour of HIBD the expression of G-CSFR was obviously promoted by G-CSF. To determine the mechanism of neuroprotective effect of G-CSF, we detected the expression of Bcl-2, Bax and Caspase3. The expression of Bcl-2 was increased while the Bax and Caspase3 were decreased significantly, which suggested that they were involved in the neuroprotective effects induced by G-CSF.4. G-CSF promoting the proliferation in neonatal mice after hypoxic-ischemic brain damageIn HIBD mice treated with G-CSF, the expression level of NSCs marker, nestin, was obviously increased in subventricular zone (SVZ), dentate gyrus (DG) and the cortex of the cerebral hemisphere, and the level of nestin was sustained in 7 days after HIBD. Brdu/nestin immunofluorescence was examined in the DG, which showed that G-CSF promoted the proliferation of NSCs. The proliferation of endogenous NSCs was abserved in the SVZ, an increase in BrdU positive cells was observed in G-CSF treated group.Conclusion:This study suggests that G-CSF can promote the viabilities of NSCs in vitro in a dose-dependant manner. The proliferation of NSCs is increased when treated by G-CSF, and the expression of p-STAT3 was up-regulated. G-CSF can promot the differentiation of neuron and neuroglial cells by regulating the genes involved in differentiation of NSCs. In the study of model of hypoxia-ischemia brain damage, the apoptosis of neuron is decreased obviously and the proliferation of NSCs in SVZ and DG was increased obviously when the mice were treated with G-CSF after HIBD, which may serve as a useful reference for future studies that aim to designing therapeutic strategies for promoting brain recovery and repair after brain damage by means fo stimulating the NSCs to proliferate.
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