| Neural stem cell (NSC) is an undifferentiated cell which is characterised self-renewal and multiple differentiation potentials in the central nervous system (CNS). Current study has shown that it can proliferate greatly in vitro and differentiate into three major types of cell (neurons, astrocytes and oligodendrocytes) in the CNS under certain inductive conditions. Yet the mechanism is unclear. Injury and repair of the spinal cord had been one of the focuses in the study of neuroscience. Two main strategies are currently considerated: one is to provide enough extrinsic neurotrophic factors for improving regenerating environment, then to promote the regeneration of the nerve. The other is cell or tissue transplantation. Embryonic brain tissue (EBT), tissue from peripheral nervous system (PNS) and some non-nervous tissue materials etc had been used. Though some improvement of regeneration had been observed, many problems are still existed.In the present study, methods including cell culture, DNA chip, gene transfection, flowcytometry, retrograde HRP tracing, in situ hybridization and immunocytochemistry etc were used in the isolation and culture of both human and rat neural stem cells, preparation of brain-derived neurotrophic facter (BDNF) genetically modified rat neural stem cells. The differentiation of influenced by multiple factors and the change of gene expression of NSC were also examined. Finally the transplatiation of NSC and BDNF genetically modified NSC to the spinal cord injury were applied . The results are as follow:1. Both human and rat embryonic brain tissues (EBT) were isolated and cultured with serum free culture medium. The cells wer collected and identifiedby nestin, also the cells were induced by 5% fetal bovine serum (FBS) and then immunohistochemistry staining for GFAP and NF-200. The results showed that the cells collected from above procedures can proliferate in vitro and differentiated multi-potentially. The recombinant adenovirus containing BDNF gene were applied to transfect the isolated rat NSC, followed by the selection with G418 and the identification by BDNF in situ hybridization, rat BDNF genetically modified NSC were successfully collected.2. FBS of series concentration were used to induce the differentiation of human NSC. The results showed: Positive ratio relation was seen between the concentration of FBS and the number of glial cells that derived from NSC.3. Microwave irradiation was effective in the proliferation, differentiation and apoptosis of human NSC and a dose-result relation had been found. After the irradiation by microwave, the cell division and the proliferation were reduced in the human NSC; The NSC were found mostly in the medium suspension with little attached to the wall of culture flask and most of them were dead. Once the NSC attached the wall, they differentiate into neurons and glial cells with a constant ratio with that before irradiation. TUNEL showed the apoptosis number of NSC by microwave increased.4. Using DNA microarray we investigated differential gene expression, comparing NSC in their neurosphere state with the cells 7 days after 10% FBS induction of differentiation. More than 1000 genes showed the differential gene expression, among them, 138 genes showed up-regulation in differentiated condition, 179 genes showed down-regulation. Genes associated with (l)cell cycle, (2)differentiation and development, (3)receptor and channel, (4) cytoskeleton, (5)intracellular signal transduction, (6)growth factor, (7)oncogene and tumor suppressor, (8)transcription and translation regulation, (9) open reading frame gene, (10)ESTs, (11 )protein transport, (12)metabolism etc.5. After the transplantiation of NSC or BDNF genetically modified NSCinto the complete transection at lumber 4 spinal cord of the rat, HE staining, X-gal staining, ISH for BDNF and GFAP, immunocytochemical staining for BDNF, GFAP and NF-200 and retrograde HRP tracing etc were applied. The results showed both NSC and BDNF genetically modified NSC we...
|
PDF Full Text Request