An Experimental Study On The Protective Effect By Adeno-Associated Virus Mediated Heme Oxygenase-1 On Spinal Cord Injury In Rats

Objectives1 To construct adeno-associated virus vector-mediated rat heme oxygenase-1 recombinant overexpression of the virus and the use of its transfected rat spinal cord tissue;.2 To investigate the protective effect of AAV-HO-1 on spinal cord injury of rats;3 To investigate the protective mechanism of AAV-HO-1 on SCI of rats.Methods1 To construct the overexpresses HO-1 recombinant adeno-associated virus vector;2 According to the different factors of the animal were divided into sham operation group(Sham group),SCI group,empty vector group(AAV-EGFP group)and AAV-HO-1 group.The SCI group,AAV-EGFP group and AAV-HO-1 group were treated with saline,AAV-EGFP and AAV-HO-1 in the local spinal cord tissue for 7 days before the establishment of spinal cord injury model.The spinal cord injury model was established by the weight compression.The motor function of hindlimb was observed by Basso Beattie Bresnahan(BBB)scores before SCI and 1d,3d,7d,14 d and 21 d after SCI.Evaluate the protective effect of over expression of HO-1 on SCI;3 Reagent kits of malondialdehyde(MDA)and superoxide dismutase(SOD)were measured to detect the changes of MDA content and SOD activity after spinal cord injury;The expressions of HO-1,Bax and Bcl-2 were detected by Western blot;The apoptosis of the spinal cord was detected by TdT-mediated UTP nick end labeling(TUNEL);The expression of fluorescence signal in spinal cord tissue was observed by fluorescence microscopy;The immunofluorescence staining was used to detect the expression of NeuN positive cells.Resμlts1 Successfully constructed HO-1 overexpressing recombinant adeno-associated virus vector;2 Successfully established a model of spinal cord injury by weight compression,The BBB scores of SCI group,AAV-EGFP group and AAV-HO-1 group were significantly lower than that of Sham group.The BBB score of AAV-HO-1 group at 7d,14 d and 21 d after SCI was significantly lower than that of SCI group and AAV-EGFP group(P <0.05);3 AAV-HO-1 group compared with SCI group and AAV-EGFP group,NeuN positive cells increased significantly(P <0.05);4 AAV-HO-1 was successfully transfected into rat spinal cord tissue and could express HO-1 at high levels consistently in spinal cord tissue;5 Compared with SCI group and AAV-EGFP group,Apoptosis index(AI)and the level of Bax protein were significantly decreased and the level of Bcl-2 protein was significantly increased(P <0.05);6 The content of MDA in AAV-HO-1 group was significantly lower than that in SCI group and AAV-EGFP group and the content of SOD in AAV-HO-1 group was significantly increased(P <0.05).Conclusions1 Overexpression of HO-1 can promote the survival of spinal cord neurons and the recovery of limb motor function in SCI rats,and has a protective effect on SCI;2 Overexpression of HO-1 can inhibit the apoptosis of rat spinal cord tissue,save the damaged spinal cord tissue,and the overexpression of HO-1 play a protective effect with the anti-apoptotic effects;3 Overexpression of HO-1 can reduce the intracellular reactive oxygen species,inhibit oxidative stress response,and the overexpression of HO-1 play a protective effect with the antioxidant effects.