| Objective: To observe the growth and differentiation of ectomesenchymal derived neural stem cells in fibrin scaffold and evaluate the curative effect of transplantation the complex of ectomesenchymal derived neural stem cells and fibrin scaffold to repair spinal cord injury in rats.Methods: 1. The ectomesenchymal stem cells(EMSCs)were obtained from nasal respiratory mucosa in vivo of adult rats,then the cells were adherent cultured repeatedly for purification. The pure EMSCs were induced into neural stem cells( NSCs) in proper conditions.2. EMSCs induced NSCs were cultured in 24-well/6-well plates with fibrin glue bedding(as the F-N group) for 2 weeks, and a control group(Control group), except without the fibrin glue bedding, the other culture conditions are the same with the F-N group, was established at the same time. Then immunofluorescence staining was used to observe the growth and differentiation of NSCs of the two groups, and Western blot was performed to analysis the expression level of NF200 and GAP43, and all comparison was made with the Control group.3. 60 Sprague-Dawley host rats were randomly assigned into three groups before T10 spinal cord transection model was established: the complex of EMSCs induced NSCs and fibrin scaffold grafting group(as the N-F group), the fibrin scaffold grafting groups(as Fg group) and the spinal cord injury group(as SCI group). Then the model rats were estimated by Basso Beattie Bresnahan(BBB) locomotor rating score every week followed respectively. The segments of spinal cord including the lesion site of the rat in the three groups were harvested and examined by immunohistochemical staining and Western blot performed for detecting the expression level of gap43, NF200 and synaptic in the lesion site.Results: 1. The EMSCs obtained from nasal respiratory mucosa cultured in vitro showed prosperous proliferation, and high expression level of CD105, CD44, nestin and vimentin when examined by immune fluorescence. EMSCs induced NSCs cultured in vitro showed prosperous proliferation and remarkable expression level of nestin and CD133 when examined by immunofluorescence.2. EMSCs induced NSCs reproduced well in fibrin glue and can differentiate into cells with neural phenotype. Long projections were emerged around the cells and the projections were well connected with each other. The immune fluorescence of the F-N group when compared with the Control group, showed that the number of cells with neural phenotype was gorgeous, and the length of the projections were longer, and the connections between the projections were more complex. Western blot showed significant difference in the expression level of NF200 and GAP43(P<0.05) between the two groups.3. The BBB scores of the N-F group were higher weekly than that of the Fg group and the SCI group begin at 2 weeks after operation and lasted to the end of the research. Motor function of Fg group also improved, but to a limited extent. SCI group almost no improvement showed in motor function when measured by BBB score. The Western blot showed that the expression levels of GAP43, NF200 and snapsin in the lesion site of the spinal cord of N-F group were higher than that in Fg group and SCI group, and the difference was statistically significant(P<0.05). The section of the lesion site of spinal cord immunohistochemistry showed that the spinal cord transection end in N-F group large numbers of immunoreactive positive nerve fibers in the form of network structure were emerged and extended into the broken end on both sides of the spinal cord parenchyma; in Fg group less fiber passed through, and no network structure was observed; in SCI group almost no positive fibers passed through.Conclusion: The EMSCs can be obtained from nasal respiratory tissue in rat, and be purified in vitro. Strong proliferations were showed in EMSCs when cultured in proper conditions. EMSCs can be induced into cells with neural stem phenotype under suitable conditions. Fibrin scaffold can promote the growth and differentiation of EMSCs from nasal respiratory mucosa induced NSCs, and it is an ideal material for spinal cord injury repair. The EMSCs induced NSCs has a significant effect on the histological reconstruction and functional recovery in SCI. |
