Study On Neural Stem Cells Characteristics Of Astrocytes

Astrocytes are the most widely distributed and most complex glial cells in the central nervous system.They constitute a functional unit with neurons and play an important role in the physiological and pathological processes of the nervous system.According to Raff's astrocytes "bispectrum theory",O-2A progenitor cells(oligodendrocyte-type-2 astrocyte progenitor)/ OPCs(oligodendrocyte precursor cells)are dual potential precursor cells,which can differentiated into both oligodendrocyts(OLs)and type-2 astrocytes(T2As).Recent,a series of studies on glial cells have found that specific microenvironment in vivo or induced in vitro can make GFAP-positive astrocytes show stem cell characteristics.In this paper,OPCs were isolated and purified by constant temperature shaking and differential adherence method,and then differentiatied into T2 As.Then we studied the changes of the biological characteristics of T2 As in the process of dedifferentiation and subsequent differentiation.Objective: The changes of biological characteristics and the expression of stem cell markers of O-2A lineage astrocytes during dedifferentiation and differentiation in vitro were studied,and further study the neural stem cells characteristics of T2 As.Methods:(1)OPCs,T1 As and T2 As that coming from the neonatal rat cortex was purified and cultured in vitro.The morphological changes of T2 As in the neural stem cell culture medium containing growth factors(EGF and bFGF)were observed,and the distinctive markers for T1As(GFAP),OPCs(A2B5)and T2As(GFAP/A2B5)were detected by immunofluorescence.(2)The expression of SOX2 and Mash1 in neural stem-like cells(NSLCs)was detected by immunofluorescence and the dedifferentiation rate of T2 A was statistically analyzed.(3)To observe the morphological changes of NSLCs in conditional medium containing low serum.Western blot was used to detect the expression of GFAP,Vimentin,SOX2,Mash1 and DCX in the process of dedifferentiation and induced differentiation of astrocyte.Neural stem cells and neuron markers in neural stem-like differentiated cells(NSLDCs)was detected by immunocytochemistry.Results:(1)OPCs,T1 As and T2 As were isolated and purified by the method of constant temperature oscillation and differential adherence method,and the cell purity was higher.T2 As were neural stem like cells in neural stem cell culture medium containing growth factor(EGF and bFGF).Immunocytochemistry showed that T1 As was GFAP positive and A2B5 negative,while OPCs were A2B5 positive and GFAP negative.T2 As labeled A2B5 and GFAP were positive.(2)The dedifferentiation rate of T2 A was(92±2.6)% by statistical method.NSLCs were SOX2 and Mash1 positive by immunofluorescence.(3)NSLCs were neuron-like morphology in conditional medium containing low serum.Western blot showed that astrocyte marker GFAP had a decreasing trend in the process of dedifferentiation and induction differentiation of T2 As,indicating that the cell has changed from glial cell type to other cell type.Vimentin was slightly expressed in T2 As,and the expression was significantly increased after dedifferentiation.In order to further demonstrate that T2 As can be dedifferentiated into neural stem cells,NSLCs were identified with recognized neural stem cell markers SOX2 and Mash1,and the results showed that the two markers were significantly higher in NSLCs compared to T2 As.These results indicate that T2 As have been transformed from astrocytes into NSLCs.The expression of SOX2 and Mash1 decreased in the cells when low serum was added to induce differentiation.The expression of DCX was significantly increased in the process of dedifferentiation and induction differentiation of T2 As,indicating that the cells began to differentiate from neural stem-like cells into neuronal cells.Neural stem cells and neuron markers in NSLDCs were positive by immunocytochemistry.Conclusions: The purity of O-2A lineage cells T2 As obtained by constant temperature oscillation method and differential adherence method was higher.T2 As can dedifferentiate into NSLCs in the culture medium containing growth factors(EGF and bFGF),and NSLCs are neural stem-like cells.The dedifferentiation rate of T2 As is about 98±1.96.NSLCs were able to induce differentiation in conditional medium containing low serum,and neuron markers were positive in NSLDCs.The neural stem cells characteristic of T2 As were further proved.