| Objective:(1) To isolate and incubate GFP+-positive neural stem cells(NSCs) derived from fetus spinal cord using free-serum cultivation method from embryonic day 16 of GFP+-pregnancy mouse and to identify GFP+-positive neural stem cells with immunofluorescence method.(2) To establish the mouse model of spinal cord injury and observe the mice motion recovery and NSCs survival, migration and differentiation after the GFP+-positive neural stem cell(NSCs) which was isolated and incubated from mouse embryonic spinal cord was transplanted into mouse models of spinal cord injury lesions. Methods:(1) The reproduction of GFP transgenic mice.(2) The spinal cord derived neural stem cells obtained from fetal mice GFP transgenic mice were isolated and cultured in vitro and identified.(3) The mice were randomly divided into control group(30), injury group(30) and cell transplanted group(30). To establish the mice model of spinal cord injury using the Impactor M-III Spinal cord impactor. The cell transplanted group was injected 5 μL NSCs suspension in the injury after striking, the damage was only striked in injury group and the control group received no treatment.(4) The BBB function scores and inclined plane test were respectively evaluated and recorded 1 w, 2 w, 4 w, 6 w, 8 w after the operation. While, six mice in each group were sacrificed. Three of six mice were using by immunohistochemical staining and Nissl staining and another three by fluorescence quantitative PCR technology to detect Nestin, ChAT and GFAP changes of gene expression and observe the mice motion recovery and NSCs survival, differentiation and injured spinal repair situation. Results:The neural stem cells were successfully cultivated in vitro. The third generation of neural stem cells were seeded onto glass slides and observed under a fluorescent microscope in a regular round, strong refraction, clear boundaries, neurospheres issued a strong green fluorescence. Nestin expression was positive using Immunofluorescence technique. The mice were exposed to hit to establish spinal cord injury model in mice using Impactor M-III spinal striker. The spinal cord of each mouse suffered a double hindlimb paralysis. BBB scores were significantly higher in the transplanted group than in injury group after the 2 w transplantation(P<0.05). The inclination angle of the swash plate were significantly higher in the transplanted group than injury group after the 4 w transplantation(P<0.05). The transplanted NSCs can survive and migrate in the body, some of which can remain undifferentiated state, a small part of which can differentiate into glia, less of which differentiate into neurons. PCR results were consistent with the results of immunohistochemical staining. Nestin was displayed at the beginning of the injection, transplanted group was significantly higher than the injury group, 2 w after the gap began to decrease and the expression was getting closer to the control group after 6 w. The ChAT expression of transplanted group and injury group were decreasing in the early damage and then began to increase at 2 w, but the ChAT expression of transplanted group was significantly higher than the injury group. The GFAP expression of transplanted group and injury group continued to rise after injury, but the expression of transplanted group was lower than injury group. The number of Nissl bodies of transplanted group and injury group continued to decline using Nissl staining after damage 1 w ~ 2 w. However, after the beginning of the emergence of new Nissl bodies on 4 w, the number of Nissl bodies in transplanted group were higher than the number of Nissl bodies in injury group. Conclusion:(1) The neural stem cells which were cultivated in vitro and transplanted to the mouse models can survive, differentiate.(2) There was a promoting effect of NSCs for the recovery of spinal cord injury. It was forming a conducive micro-environment to resume by transplanting cell and increasing the proportion of neurons to promote recovery. |
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