| Objective: 1.Establishing the rat spinal cord hemi-section injury model.2.To investigate the effect of Bo NT/A HC the expression of the local proteins after spinal cord injury in rats.3.To investigate the effects of Bo NT/A HC on the expression of selective growth-associated proteins GAP 43 and SCG 10 after spinal cord injury.4.To investigate the effects of Bo NT/A HC on the neurite outgrowth after spinal cord injury.5.To investigate the effects of Bo NT/A HC on the recovery of hindlimb function after spinal cord injury.Methods: 1.An unilateral(left)lumbar spinal injury was achieved by nipping against the left side of the dorsal median vein with the tip of a pair of fine forceps.2.1 cm segment of spinal cord containing the lesion center was dissected out out in the control group,SCI group(2d),SCI+Bo NT/A HC treatment group(2d)and then used for two-dimensional electrophoresis and silver nitrate staining.3.1 cm segment of spinal cord containing the lesion center was dissected out in the control group,SCI group(2d),SCI+Bo NT/A HC treatment group(2d)were collected for the proteomics investigation by using isobaric tags for relative and absolute quantitation(i TRAQ)combined with two dimensional-liquid chromatography-mass spectrometry-mass spectrometry(2D-LC-MS/MS)and bioinformatic analysis.4.Based on 2D gel and i TRAQ proteomic analysis,selective growth associated proteins(GAP 43,SCG 10)were selected to be the representative as the target proteins of Bo NT/A HC.Their expression and distribution were detected in all groups [control groups,SCI groups(2d,7d,14 d,28d)and SCI+Bo NT/A HC groups(2d,7d,14 d,28d)] by western blotting and immunofluorescence.5.The positive immunofluorescence staining of SCG 10 was applied to measure of neuronal processes.The measurement of neuronal process included the length of neuronal processes,the number of neurites and the percentage of cells with neurites to total calculated SCG 10 positive cells.6.Meanwhile,the alterations in sensory and motor function at the ipsilateral hindlimb of rats were evaluated by the thermal hyperalgesia test and the grip power test,respectively.Results: 1.Both motor and sensory function appeared remarkably impaired in SCI rats.2.The results from 2-D gel plus nitrate silver stain showed that the administration of Bo NT/A HC based on SCI altered the spinal protein expression.The decrease or increase in the expression of some protein dots /dots group was clearly seen after single SCI.However,these changes were transformed by Bo NT/A HC treatment,which appeared as a reversed pattern turning toward that in control group or further increased expression upon SCI.Particularly,MW was located between 35-45 k Da and 18-25 k Da,and the isoelectric point was in the range of 4-7.3.2864 differentially expressed proteins(DEPs)were identified using i TRAQ-labeling technology,and dynamic proteins were 477.Among these proteins,146 DEPs were identified between the SCI group(2d)and the control group,including 92 up-regulated and 54 down-regulated.300 proteins were differentially expressed between the SCI+Bo NT/A HC group(2d)and the control group,including 199 up-regulated and 101 down-regulated.188 proteins were differentially expressed between the SCI+Bo NT/A HC group(2d)and the SCI group(2d),including 110 up-regulated and 78 down-regulated.Bioinformatics analysis showed that some of these proteins were associated with nerve regeneration after SCI,among these proteins,GAP-43,Apo E,S100,heat shock protein,glial fibrillary acidic protein (GFAP),myelin Oligodendrocyte glycoprotein(OMgp)were some proteins that closely involved in neural regeneration and neuroprotection.4.Western blotting results suggest that the administration of Bo NT/A HC to the SCI significantly amplified the expression of phosphorylated GAP-43(p-GAP43)and SCG 10(P<0.05).Immunofluorescence results show the positive immunofluorescent staining for both p-GAP 43 and SCG 10 was mainly distributed nearby the proximal area of injury,both cytoplasm and neuronal processes were appeared positively stained.In addition,the positive p-GAP 43 and SCG 10 expression was mainly within the cell body in the SCI-only groups.5.The measurement of neurite outgrowth based on the immunofluorescence staining of SCG 10 suggested that the outgrowth of neurites were stimulated by the Bo NT/A HC treatment;this was evident from the increase in neurite length,number of branches and the percentage of cells with neuronal processes.6.The results from the spinal function tests suggested that the Bo NT/A HC did not affect sensation,but had a large role in improving the ipsilateral hindlimb grasp power(P < 0.05).Conclusions: 1.Using two-dimensional electrophoresis and i TRAQ technology-dependent proteomics,we found that there were a large number of differentially expressed proteins in the spinal cord tissue of rats exposed to Bo NT/A HC compared with normal spinal cord and the injuried spinal cord,some of which may be related to nerve regeneration and neuroprotection.2.Bo NT/A HC to rats with SCI increased the local expression of p-GAP 43 and SCG 10,which might be affiliate the regeneration of neuronal processes surrounding the injury,probably also be favorable to the relief of spinal motor dysfunction. |
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