Experimental Study On Differentiation Of Neural Stem Cells Induced By C6 Glioma Cells

Object:Neural stem cells(NSCs) are a class of multipotent stem cells capable of producing neurons,astrocytes and oligodendrocytes in appropriate micro-enviroment, which are special cells population during nervous system development.We aimed at establishing a stable,standardized culture system of NSCs,exploring the effects of NSCs differentiation induced by C6 glioma cells on the foundation of culture in vitro, and conducting a preliminary exploration of the mechanism.This not merely enriches understanding in the interaction between the glioma cells and NSCs,but is an alert in the clinical treatment of stem cells with safety.This study will lay the experiment foundation for the differentiation and regulation mechanism of NSCs.Methods:The NSCs were derived from the striatum tissues of rat embro at the age of 17-18 days and proliferated steadily under the effect of epidermal growth factor(EGF) and B27.The rat NSCs and the differentiation NSCs were identified respectively by immunofluorescence staining including Nestin,β-tubulinⅢ,glial fibrillary acidic protein(GFAP) and oligodendrocytes marker O4.C6 glioma cells were cultured in RPMI1640 contained 10%FBS,when cells growed at 80%degree of infusion,the medium was changed to RPMI1640 without serum,24h later,the culture supernatant of C6 glioma cells were prepared.The NSCs were divided into two groups:①control group:culture with NB/RPMI1640(1:1,without EGF)②treated group:culture with NB/C6 culture supernatant(1:1,without EGF).Differentiation of NSCs was observed with light microscope at different time(1d,3d,5d).RT-PCR, Western-blotting and immunofluorescence assay were used to detect expression levels of microtubule-associated protein-2(MAP-2).Meantime,transcription regulation factors including Ngn3,NeuroD and NeuroD2 were examined at mRNA level by RT-PCR.Results:The cultured NSCs proliferated,forming neurospheres.The rat NSCs after the culture and passage were proved by positive Nestin staining.The differentiated cells in the sphere could express specific antigens of neurons(positiveβ-tubulinⅢ), astrocytes(positive GFAP) and oligodendrocytes(positive O4).The MAP-2 expression level of NSCs were observed respectively at 1d,3d and 5d.Compared with the control group,there was no obvious difference at the 1d,the MAP-2 mRNA expression level showed difference at the 3d,and the significant difference was observed both mRNA and protein level.And also,immunofluorescence staining showed more MAP-2 positive cells.Simultaneously,we detected the mRNA expression levels of transcription factors such as Ngn3,NeuroD and NeuroD2 with the same trend.Conclusions:This study successfully established the culture of rat NSCs in vitro,that can maintain a high degree of proliferation and non-self-differentiation of NSCs. Compared with the control group,C6 glioma cell culture supernatant can induce NSCs to differentiate into more neurons,which are probably by upregulating the expression of transcription factors including Ngn3,NeuroD and NeuroD2.We supposed that C6 glioma cells may secrete some factors inducing NSCs to differentiate into neurons through regulation of the bHLH family of transcription factors.It plays foundation for further study of NSCs differentiation and relations between that and the origin of glioma.