| The morbidity rate of spinal cord injury(SCI) increased annually with great harm, and neurological recovery is limited, which has been regarded as an important medical topic and public issue. Currently, the neural stem cells(NSCs) transplantation has become a new treatment strategy for SCI. However, NSCs transplantation alone does not show satisfactory effect as expected for SCI, mainly blame to insufficient differentiation to functional neurons instead of glial cells. Thus combination strategy of NSCs with neurotrophins sheds light on the repair of SCI. As a neurotrophic factor, NT-3 plays an important regulatory role in survival, proliferation and differentiation of nerve cells upon binding to its specific receptor tyrosine kinase receptor C(Trk C). In addition, Transcription factor SRY(sex determining region Y)-box 2(Sox2), plays its roles not only in the maintenance of stemness but also in the differentiation of nerve cells which has been drawn much attention. Through a series of experiments, the present study revealed that NT-3 regulates the expression of Sox2 in NSCs, which in turn skews the differentiation of NSCs to neurons. In addition, Sox2 may regulate the expression of the Trk C protein by regulating its gene transcription, thereby making an effect on the differentiation of NSCs into the direction of neurons.Materials and Methods1. Isolation and identification of primary NSCs in ratsNSCs were isolated from Sprague-Dawley(SD) rat embryos(14 ~ 15 days) and cultured in vitro by using the method of Pluchino. Firstly, in order to detected NSCs specific markers, neurospheres were placed in confocal dishes coated with Poly-L-ornithine(P-L-O) and cultured for 24 hours(h), then the expressions of Nestin and Sox2 were assessed by immunofluorescence(IF) assay. In addition, for the purpose of verified its pluripotent, after the cells were treated for 48 h with differentiation medium---with 1% fetal bovine serum(FBS) instead of growth factors, the expressions of neuronal precursor cell marker doublecortin(DCX), oligodendrocyte transcription factor 2(Olig2) and glial fibrillary acidic protein(GFAP) were assessed by IF assay.2. The impact of NT-3 on Sox2 expression in NSCsTo explore the effects of exogenous NT-3 on Sox2 expression in NSCs, primary cultured NSCs were randomly divided into: control group, 25 ng/ml NT-3 group, 50 ng/ml NT-3 group, 100 ng/ml NT-3 group. After the cells in all groups were treated for 24 h, we detected the protein expression of Sox2 by Western blotting.3. Effects of NT-3 on NSCs differentiationGrouping as above, we further observed the effects of exogenous NT-3 on NSCs differentiation. After the cells were treated with different concentrations of NT-3 for 24 h, the expressions of DCX, GFAP, Olig2 were assessed by Western blotting and IF assay.4. NT-3 regulated Sox2 through PI3K/Akt signal pathwayIn order to verify the PI3K/Akt signal pathway in the role of NT-3 regulating Sox2, primary cultured NSCs were randomly divided into: control group, 10 u M PI3K/Akt pathway inhibitor(LY294002) Group, 100 ng/ml NT-3 group, LY294002(10 u M) + NT-3(100 ng/ml) group. After the cells in each group were cultured for 24 h, we assessed the protein expression of phosphorylated Akt(p-Akt), Sox2 and DCX by Western blotting.5. Statistical analysisAll quantitative data were presented as means ± standard deviation. Statistical analysis was performed using the one-way analysis of variance(ANOVA) test. A value of p<0.05 was considered statistically significant.Results1. Neural stem cells with multiple differentiation potential were successfully purified and cultured.Firstly, cells harvested from SD embryonic brain were cultured for 3 days and typical shape of NSCs were observed with phase contrast microscopy. The nature of cultured primary NSCs were also determined by immunofluorescence staining of NSCs specific marker Nestin and stemness marker Sox2. The multiple differentiation potential of NSCs was corroborated by staining mature neural cell lines, specifically DCX for neuronal precursor cells, Olig2 for oligodendrocyte and GFAP for astrocyte marker.2. NT-3 promoted Sox2 expression in NSCs.We observed that NT-3 showed a dose-dependent effect on Sox2 expression in NSCs. Compared with the control group, higher concentration of NT-3(25 ng/ml, 50 ng/ml, 100 ng/ml) upregulated Sox2 protein expression at a significantly higher level in NSCs(P<0.05).3. Upregulated Sox2 resulting in skewed differentiation to neurons of NSCs.Firstly, compared with the control group, higher concentration of NT-3 upregulated Sox2 protein expression at a significantly higher level in NSCs(P<0.05). Western blotting also showed that with the increasing concentration of NT-3, DCX expression levels are significantly increased compared with the control group, the difference was statistically significant(P<0.05); In addition, 50 ng/ml NT-3 group and 100 ng/ml NT-3 group also promoted Olig2 expression(P<0.05). Secondly, NT-3 significantly skewed the differentiation of NSCs to DCX positive(+) cells also in a dose-dependent manner detected with IF assay. DCX + cells was significantly increased in 50 ng/ml NT-3 group and 100 ng/ml NT-3 group(P<0.05). Increased Olig2 + cells percentage also was observed in 50 ng/ml NT-3 group and 100 ng/ml NT-3 group compared with the control group(P<0.05).4. PI3K/Akt signal pathway mediated NT-3-induced upregulation of Sox2.The expression of phosphorylated Akt(p-Akt) was significantly upregulated in NT-3 treated NSCs with the highest upregulation of p-Akt in 100 ng/ml NT-3 group, correlated with the upregulation of Sox2. To confirm the specificity, a specific inhibitor of PI3K/Akt signal pathway, LY294002(10 u M), was used to block NT-3-induced Sox2 expression. LY294002 significantly decreased Akt phosphorylation and subsequently decreased Sox2 expression in NT-3-treated NSCs(P<0.05) showing NT-3 upregulates Sox2 expression through PI3K/Akt signal pathway.Conclusions1. Exogenous NT-3 promotes Sox2 expression in a dose-dependent manner and skews the differentiation of neural stem cells to neuronal precursor cells and oligodendrocytes.2.PI3K/Akt signal pathway plays an important role in NT-3 induced Sox2upregulation and skewed differentiation of NSCs.DMEM/F-12 100 ml FBS 1 ml(6)Zamboni固定液... |
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