| The characteristics of neural stem cells NSCs are:NSCs can self-renewal and proliferate; NSCs can be differentiated into neurons and neuroglia; NSCs have low original immunity; NSCs can be cultured for long time.The functions of NSCs are:NSCs can repair the injury of brain nervous system; NSCs can be used for gene therapy of nervous system diseases as carriers.NSCs reside in different regions of central nervous system (CNS) of mammal. Since the percentage of brain of neonate rat is bigger than that of the adult and NSCs are concentrated, we choose the main regions of NSCs from hippocampus of neonate rats to obtain more NSCs easily.Many cell factors secreting from astrocytes can promote the differentiation of NSCs into neurons. These factors play an important role in the normal movement and substance metabolism of neuron. It is useful for recognizing biological function of astrocytes gained from the cerebral cortex of neonate rats, which is the connective location between neurons and astrocytes.NSCs are ideal donor cells to treat nervous system disease; Neurons differentiated from NSCs play the main therapeutic action. Differentiation was one of the important properties of NSCs. It was influenced by numerous factors and depends much on neurotrophic factors in micro-environment.ObjectiveTo investigate the effect of astrocytes on the differentiation of NSCs isolated from hippocampus of neonate rat into neurons. Materials and Methods1. Materials12 Wistar rats of neonate rats2. Methods(1) The culture and identification of NSCsThe DMEM/F12 media containing basic fibroblast growth factor (bFGF). epidermal growth factor(EGF) and B27 were used to cultivate NSCs isolated from hippocampus of 2-3d Wistar rats. Cell suspension was prepared and diluted when the diameters of the third-passaged cell spheres were 200μm. Monoclone was done by utilizing limiting dilution assay. Monoclonal cells were passaged and divided into two parts and experimented respectively as follows:Immuno fluorescence staining for Nestin was used to identify monoclonal NSCs for one part and the other was induced and differentiated by DMEM/F12 containing 10% FBS for 5 days. Immunofluorescence staining for glial fibrillary acidic protein (GFAP). neurons specific enolase (NSE) and galactocerebroside (Galc) were used to identify astrocytes. neurons and oligodendrocytes. respectively.(2) Collecting ACMThe astrocytes separated from the cerebral cortex of 2-3d Wistar rats were cultivated in the DMEM/F12 (1:1) media containing 15% FBS. Astrocytes were purified by the differential speed and shaking at room temperature.Immunofluorescence staining for GFAP was done to part of the third-passaged cells, and the purity of astrocytes was labeled. The other astrocytes were cultivated in the DMEM/F12 media of NSCs for 48h, then the astrocytes-conditioned medium (ACM) were collected.(3) The samples of neural stem cells were divided into groupsMonoclonal cultured NSCs were inoculated in six well plate (2 holes each), grouped as follows:Group A:Control group;Group B:ACM:NSCs (1:2) group:Group C: Astrocytes and NSCs transwell co-culture group.Immunofluorescence staining and western blot assays were done to cells a week later to detect the percentage of neurons and the expression of NSE.(4) Statistical analysis The statistical software SPSS 13.0 was applied for data analysis forthe percentage of neurons, mean±standard deviation (x±s) was used for all data, T-test was used between group and group, P<0.05 was significant in statisticResults1. Identification of NSCsThe cultured monoclonal spheres expressed Nestin, and expressed NSE, GFAP and GalC 5d after induced differentiation by immunofluorescence stainning.2. Identification of astroglia96.5% GFAP positive cells were found by immunofluorescence stainning. The purity of GFAP can be used to following tests.3. Differentiation results of NSCs into neuronsThere was difference of differentiation speed among group A, group B and group C. Group B, group C grew faster than group A 3 days after induction and differentiation. The percentages of NSE positive cells of group A, group B and group C were 14.7%±3.5%,35.2%±4.1 and 40.3%±3.7 respectively 7 days after induction. The proportions of neurons in group B and C were much higher than group A (P<0.05), and the highest was group C. There is no significant difference between group B and C (P>0.05) Western blot indicated that there was significant increase in group B and group C, and no significant difference between the two groups.Conclusionl.Astrocytes can promote the differentiation of NSCs from hippocampus of neonate rats into neurons.2.Effect of Astrocytes on the differentiation of Neural Stem Cells is related to their active substances...
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