An Experimental Study On The Therapeutic Effect Of RAAV2-Mediated HBDNF Gene Combined With Swimming Training After Spinal Cord Injury In Rats

Objective:1、To construct recombinant adeno-associated virus-2（rAAV2-BDNF–GFP） that express green fluorescent protein and BDNF protein.2、Gene therapycombined with swimming training after spinal cord injury in rats,through the analysisand evaluation of its behavior、histology and Immunohistochemistry, and to comparewith single intervention methods, explore the protective effect and impact of genetherapy combined with swimming rehabilitation after spinal cord injury in rats.Methods:1、we used the original plasmid pTRGDNFGFP as template，constructedrecombinant plasmid pTRBDNFGFP that contains hBDNF cDNA fragments. Then thepackaging cell lines(HEK293T cell) were co-transfected with the pTRBDNFGFPtogether with the control plasmid pAAV-RC and pHpelper by the PEI method, producedrecombinant adeno-associated virus-2that express BDNF.2、 The packagedrecombinant adeno-associated virus-2was purified with three steps method ofChloroform treatment-PEG/Nacl precipitation-chloroform extraction. We usedSDS-PAGE method to detect virus purity， and used the real-time fluorescentquantitative PCR detect virus titer. After rAAV2-BDNF-GFP Infected HEK293cell，weused Western Blot detect cell BDNF protein expression.3、90healthy adult male SDrats were randomly divided into5groups：swimming training group(group A,n=18),pure gene therapy group (group B, n=18), gene therapy combined with swimmingtraining group (group C, n=18), sham group(group D,n=18), natural healinggroup(group E,n=18). Sham group rats knock out Lamina, exposed spinal cord. The restof the four groups used modified Allen ’s strike method manufacturing rat spinal cordinjury model.4、Hind limb function score of the experimental rats will be tested1dbefore modeling and after modeling for1d、3d、7d、14d、28d、42d. Spinal cord tissue was removed at different time points（at day1、3、7、14、28、42），Each group has6rats. Then，HE staining and immunohistochemical staining.5、Immunohistochemicalstaining slices was analyzed and processed by Image-Pro plus6.0. Statistical data wasanalysed by statistical software SPSS17.0.Results:1、Successfully constructed a recombinant plasmid pTRBDNFGFP; Afterthe rAAV2-BDNF-GFP were constructed and purified and condensed, the titer ofrAAV2-BDNF-GFP was6.83×1011vp/ml. After recombinant virus infected HEK293Tcells, green fluorescence can be observed under a fluorescence microscope; WesternBlot proved that infection HEK293cells infected by the recombinant virus has theability to express BDNF.2、BBB score and inclined plate experimental results show thatat different time points（at day14、28、42），compared to group A(P＜0.05)、B(P＜0.05)、E(P＜0.01)，the result of group E are significantly improved, compared to groupE（P＜0.05）,the result of group A、B are improved. The experimental results of Dgroup had no statistical significance; The results of HE staining showed that the syrinxcavity and spinal cord scar of group C less than others. The results of IHC showed thatthe AOD of group C higher than the other group(P＜0.05).Conclusion:1、Successfully constructed recombinant adeno-associated virus-2（rAAV2-BDNF-GFP）.2、rAAV2-BDNF-GFP can infect spinal cord tissue and canexpress BDNF.3、Reasonable swimming training and gene therapy can improve hindlimb motor function of spinal cord injury in rats.4、Therapeutic effect of group E wassignificantly better than the other group.5、Spinal cord injury is a very complicatedpathologic process, relying solely on a single treatment is very difficult to achieve idealtreatment effect, so the combination therapy should become a trend in the treatment ofspinal cord injury.