| ObjectiveI. To explore the feasibility of culture and identification of neural stem cells(NSCs) which come from ependyma, subventricular zone and cotex of fetal and adult rat respectively, in order to make the basis for treatment of the degeneration disease by NSCs transplantation, and to ?offer some parameters for the isolation and differentiation of NSCs derived from other kinds of tissue.II. To estimate the effective of immunomagnetic beads(d^50 run and d^= 450 nm) technique following the separation of the neural stem cells from the fetal/adult rat brain.During the estimation,some experimental methods including morphology, immunocytochemistry etc. were performed.ID. To identify the properties of the differentiated cells by immunocytochemistry, immunofluorescence and RT-PCR assays in order to check the expression of neural associated genes.Methods and ResultsI. All the seed cells derived from ependyma, subventricular zone and cotex which were isolated from both fetal and adult SD rat respectively. And then they were cultured, differented and subcultured continuously in a "NSCs culture medium", identificated by flow cytometry (FCM) andimmuncytochemical Nestin, NSE and GFAP antibodies for the differenced NSCs, neuron-like cells and neuroglials-like cells respectively.There was a quick proliferation in the four histocytes under some corresponding conditions, and a "neurologic sphere" to be formed, which made up of many cells and expressed Nestin antigen. After continuous culture and subculture, NSCs might divise and proliferate again. Some buds developed into apophysis and formed nerve fiber-like structrue further, while the soma enlarged the cells with "long apophysis", which connected and crisscrossed with each other, and might be confirmed as the neurons and neurogilials respectively after immunocytochemistry identification. In the four of histocytes, fetal rat might own more NSCs than adult rat. On the other hand, the NSCs showed more in ependyma and subventricular zone than in cortex. There were no special difference in morphology or differentiated degree between ependyma (or subventricular zone) NSCs and cotex NSCs.II. Cell suspension of the fetal rat cerebral cortex was at first incubated with antibody of Nestin, and then these labeled Nestin-positive cells were separated from the suspension through the magnetic field where immunomagnetic beads coated with the second antibody existed. The purity and vitality of the separated cells were determined. Then these cells were in vitro dissociated with serum of fetal bovine for 3 days. Binding of the sorted cells to beads was shown under scanning electronmicroscope. Immunocytochemistry was performed to identify the properties of the differentiated cells. The results showed the purity of the separated cells as (89.2±4.8)%, and the vitality of the cells as 95%. The treated cells expressed neural proteins such as NSE and GFAP.III. Immunocytochemistry and immunofluorescence were performed to identify the properties of the differentiated cells.The expression of neural associated genes were detected in mRNA level by RT-PCR assays.ConclusionsI. NSCs exist not only in ependyma and cotex of SD fetal rat, but also in the subventricular zone and cotex of SD adult rat. Fetal rat nerve tissue-6-possesses much more the NSCs than adult one.II. The immunomagnetic cells sorting system we used can effectively separate neural stem cells from the cerebral cortex cell suspension and maintain the vitality of the cells well. The sorted cells might be considered to provide for the subsequent experiments.III. The NSCs separated from SD rat nerve tissue had the abilities of proliferation and multi-differentiation.The induced cells might express some neural associated genes, which implied the separated cells belonged to the NSCs of central nervous system.
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