| Objective: In recent years,stem cell/tissue engineering scaffold transplantation has attracted extensive attention in the research of spinal cord injury repair.Based on the ability of neural stem cells to self-renew and differentiate into neurons,astrocytes,and oligodendrocytes under disease-damage conditions,it plays a key role in promoting the repair of spinal cord injury.The purpose of this study is to explore:(1)To study the effects of fibrin scaffold that can release recombinant SHH adenovirus slowly on the proliferation and differentiation of neural stem cells in vitro(NSCs).(2)Construction of rat spinal cord injury(SCI)animal model,the effects of FG-SHH sustained-release scaffolds on the biological characteristics of endogenous NSCs were observed and evaluate its effect on the repair of spinal cord injury in rats.Method:(1)NSCs were isolated and cultured in vitro by suspension culture,and their surface markers were identified by immunofluorescence assay.(2)Recombinant adenovirus was used as the carrier of SHH gene,injecting it into fibrin glue scaffolds at appropriate concentration to construct sustainedrelease fibrin glue scaffolds,and its microstructure was observed under scanning electron microscope,detection of sustained release of scaffold by immunofluorescence and western blotting.(3)To observe the effect of the constructed sustained-release scaffold on the proliferation and differentiation of NSCs;Experimental group: NSCs were planted in recombinant SHH adenovirus-scaffold group(FG-SHH),culture plate,recombinant SHH adenovirus transfection group(SHH),fibrin scaffold group(FG)as control group(Controls).The effect of stent on proliferation of NSCs by MTT assay;Immunofluorescence and western blotting were used to detect the expression of β-tubulin III,MBP,GFAP and Synapsin.(4)Sixty healthy SD rats were selected to establish spinal cord injury model and random Ly divided into five groups:(A)simple spinal cord transection injury group;(B)recombinant SHH adenovirus injection group(SHH);(C)simple FG scaffold transplantation group;(D)FG-SHH scaffold transplantation group.BBB scores were used to evaluate the function of the hind limbs in each group;Spinal cord tissues were taken out at 12 weeks after operation;The expression of β-tubulinⅢ、GAP43、NF200、MBP、 GFAP and other nerve fiber junction regeneration and protein were detected by western blotting and immunohistochemical staining.Result:(1)The expression of nestin and sox-2 was high in NSCs cultured in vitro.(2)The FG-SHH scaffold showed a three-dimensional spongy reticular structure under scanning electron microscopy,it has a good sustained release effect on recombinant SHH adenovirus and can be maintained for about 6 days.(3)NSCs could stably express SHH after transfection with recombinant SHH adenovirus.(4)FG-SHH scaffolds had a strong ability to promote the proliferation of NSCs,and the positive cell rates and protein expressions of β-tubulin III 、MBP and Synapsin in FG-SHH group were higher than those in control group(p < 0.05),while the positive cell rates and protein expressions of GFAP in FGSHH group were lower than those in control group(p < 0.05).(5)BBB scores in FG-SHH scaffold group increased significantly from the seventh week after surgery compared with control groups.(P <0.05);(6)The results of immunohistochemical staining and western blotting showed that the expression of β-tubulinⅢ、NF200、GAP43 and MBP protein increased and the expression of GFAP decreased at the two ends of the spinal cord injury in the FG-SHH stent group.Conclusion:(1)FG-SHH scaffolds can promote NSC proliferation and differentiation into neurons and oligodendrocytes and inhibit the differentiation of astrocytes.(2)FG-SHH scaffolds can recruit endogenous NSCs and promoting its differentiation into neron-like cells,reduce the formation of glial scar and improving local microenvironment... |
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