| Neural stem cells(NSCs) are special cells which have some capacity for self-renewal and the potencies to differentiate with multi-direction. The discovery of neural stem cells is a breakthrough for neural regeneration in the central nervous system(CNS). We may use it to explore the molecular mechanism of development of nervous system, or as a cell resource for cell substitute therapy and gene carrier for gene therapy to treat CNS trauma, degeneration diseases of CNS and brain tumor, etc. Establishing a stable and reliable culture model of NSCs in vitro is a prerequisite to explore the mechanism of its proliferation or differentiation and the transplantation in vivo. It has become a hot topic in neuroscience. Nitric oxide (NO) is an important signal molecule in the process of neural development and regeneration. It plays an important role not only in the signal conduction as a neurotransmitter and second messenger, but also in the regulation of neurite growth, synapse establishing, neural migration, cell proliferation and differentiation. So the research of the relationship between NO and NSCs is significant to discover the effect of NO on neural development and regeneration. It is helpful to illuminate the mechanism ofproliferation and differentiation in NSCs. Since NO is very unstable and difficult to be examined directly, indirect method is often used by examining the nitric oxide synthase (NOS). Nitro-compound such as sodium nitroprusside (SNP) is used as donor of ectogenic NO in the study.The first part of the experiment was to isolate the stem cells from the mice cortex in medium containing EGF. The primary culture cells had the ability to consecutively proliferate and form neurosphere in the medium. Nestin was positively expressing in these cells which were examined by immunohistochemistry. The cells could differentiate into adult neural cells in medium supplemented with fetal bovine serum. The expression of NF-200 were positive in some of the differentiated cells and some of them were GAFP positive. It demonstrated that some of the differentiated cells are neurons and the others were glial cells. These results showed that the primary culture cells were NSCs.The expression of nNOS, eNOS and iNOS in NSCs were examined by immunohistochemistry in the second part of this experiment. The expressions of NOS in NSCs were compared between normal condition and hypoxia. We found that nNOS and eNOS were positive in NSCs which were cultured at general condition, while the iNOS was negative. After exposed to acute hypoxia for 10 minutes and 30 minutes, the expressions of nNOS , eNOS and iNOS are positive. The intensity of iNOS expressed in the cells is correlated positively with the time of hypoxia.SNP was used as a donor of NO with different concentration in the third part of this experiment to study the inhibiting effect on NSCs. The mediums of the experiment groups are supplemented with SNP at the concentration of 100μmol·L-1, 150μmol·L-1 and 200 μ mol · L-1, and the control group was non-SNP. The release of NO in the medium from SNP was measured by the Griess assay. Half of the cells in the culture holes were measured by MTT assay to reflect the amount of living cells after 24 hours, the control group was also correspondingly measured by MTT assay. The NSCs which were affected by SNP could be induced to differentiate in medium supplemented with fetal bovine serum. The expression of NOS in NSCs was examined by immunohistochemistry. The release of NO was increasing along with the enhancement of the concentration of SNP; The amount of living cells in experiment groups was decreased comparing with the controls and the level of decrease was dependent on the concentration of SNP; The NSCs still had the ability to proliferate actively and could be induced to differentiate into neuron and glial cell in medium when SNP was removed. We found that SNP could promote the differentiation of NSCs. The expressions of nNOS and eNOS were positive in NSCs which were affected by SNP, but the expression of iNOS...
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