Identification, Differentiation And Expression Of GM-CSF MRNA Of Neural Stem Cells From Dentate Gyrus Of Hippocampus In Newborn Rats

Neural stem cells (NSCs) are some cells that not only can maintain the capacity for self-renewal and provide plenty of cells of the CNS but also can differentiate into neurons, astrocytes or oligodendrocytes. Generally speaking, NSCs have three kinds of properties at least. Firstly, they are deficient in mature symbols in the nervous system, such as neuron specific enolase(NSE) or glial fibrillary acidic protein(GFAP). But they give expression to nestin and receptors of epidermal growth factor(EGF) and basic fibroblast growth factor(bFGF); Secondly, they possess the capacity for self-renewal; Thirdly, they possess the capacity for mul tipotential ity, namely differentiating into various cells in the central nervous system. The discovering of NSCs provides a possibility for the therapy of neural degeneration diseases. So far, the NSCs having the capacity for multipotential differentiation have been isolated and cultivated successfully from the umbilical blood, partial regions of embryo or adult mammalian brain. However, the number of NSCs in adult mammalian brain islimited, and the procedure of culturing NSCs from embryo mammalian brain is complicated. It has been not reported whether the NSCs can be isolated and cultured from the dentate gyrus of hippocampus of newborn rat or not so far.Studies indicated that some cytokines might be related to the proliferation and differentiation of the neural stem cells, and could affect the whole process of differentiation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) exists widely in the hematopoietic system, immune system and nervous system. Evidences showed that it contributed to proliferation and differentiation of many kinds of cells, and GM-CSF also improved the livability of neuron in vitro and reduced its apoptosis rate. But the understanding of effects of GM-CSF on NSCs is still poor. EXPERIMENTAL PRECEDURE1 Immunohistochemistry was applied to detect nestin-positive cells in dentate gyrus of hippocampus of rats at the first day after birth. Continuous passage of neurospheres was used to isolate, culture NSCs from dentate gyrus of hippocampus of newborn rats and to observe the self-renewal of neurospheres, which were immunostained with nestin.2 MTT Assay was used to examine OD value of Terasaki wells . OD value was measured once a day for 8 days and mapped growing curve of cells to determine the activation of proliferation in primary cells.3 After incubated with 5-bromo-2-deoxyuridine(BrdU) for 7 days, newborn cells derived from regenerative neurospheres were immunostained.4 The cells were collected by centrifugation separation and put into poly-1-lysine-coated Terasaki wells, which were randomly divided into one of three groups of 24 wells cells each. Group I contained DMEM/F12 serum-free medium with 10% calf serum; Group II contained DMEM/F12 serum-free medium with 10% calf serum and 100ng/mlGM-CSF; Group III contained DMEM/F12 serum-free medium with 100ng/mlGM-CSF. After successively cultured with 5% CO, at 37癈 for 7 days, the cells were respectively stained with cell-type-specific markers, i.e. NSE for neurons and GFAP for astrocytes.5 The expression of GM-CSF mRNA in the NSCs was detected by in situ hybridization.All the data were quoted as mean ±SD and statistical analysis were performed using Student's t test, Chi-Squre test and Correlations assay. The criterion for statistical significance was P<0.05. RESULTS1 Abundant nestin-positive cells were found in the dentate gyrus of newborn rats. Cells isolated from dentate gyrus of hippocampus divided and proliferated into neurospheres 5-7 days after the culture, in passage experiments, the neurospheres were collected by centrifugation separation and dissociated into single-cell suspension. These single cells came into being regenerative neurospheres in medium.2 After incubated with BrdU for 7 days, the newborn cells became BrdU-positive. OD value of Terasaki wells declined slightly on the thirdday. OD value of Terasaki wells increase...