Therapeutic Benefit Of Intravenous Administration Of Adenoviral Vector Mediated HBDNF Gene-modified RMSCs After Spinal Cord Injury In Adult Rats

Part OneObjectives To construct the recombinant adenovirus vector carrying human BDNF marked enhanced green fluorescent protein(EGFP).Methods The hBDNF gene was constructed by PCR with plasmid pDC316-hBDNF as template .With enzyme digestion, the hBDNF gene was inserted into the vector pDC316-mCMV-EGFP and the shuttle plasmid pDC316-hBDNF-mCMV-EGFP was constructed, which was cotransfected with the adenovirus skeleton plasmid pBHGlox_E1,3Cre into 293 cells to obtain the produced replication defective recombinant adenovirus vector Ad-hBDNF-EGFP. The recombinant adenovirus was propagated by repeat infection of 293 cells and purified by ion exchange method , then the virus particles were counted and the purity and titer were determined. Results Both recombinant shuttle plasmid pDC316-hBDNF-mCMV-EGFP and recombinant adenovirus vector Ad-hBDNF-EGFP were successfully constructed identified by PCR amplification, restriction analysis and sequencing. After propagation and purification , the virus particle count , OD260/ OD280 and titer of recombinant adenovirus were2.4×1011VP/ ml , 2.0 and 0.8×1010CCID50/ ml respectively.Conclusions Recombinant adenovirus vector Ad-hBDNF-EGFP was successfully constructed ,which laid a foundation of further study on infection of rMSCs and gene therapy of SCI. green fluorescent protein(EGFP); Genetic recombinationPart TwoObjectives Recombinant adenovirus vector Ad-hBDNF-EGFP transfect the rat mesenchymal stem cells (rMSCs) to get hBDNF gene-modified rMSCs. Methods rMSCs were separated from the bone marrow of SD rats, cultured in vitro, To detect the surface marker antigens with FACScan flow cytomete (FCM) when the rMSCs proliaferaterd into the third dgeneration. The third generation rMSCs were infected by the recombinant adenovirus vector Ad-hBDNF-EGFP constructed formerly and identified by fluorescent microscope. And the rate of transfection was analysed with flow cytometer. The level of hBDNF mRNA expression were observed by RT-PCR and the level of hBDNF expression were examined by Western blotting. Results rMSCs were successfully separated, cultured and propagated. The rMSCs of the third generation were detected by FCM. rMSCs were uniformly postive for CD29,CD90, with negative expression of CD34,CD45. After the recombinant adenovirus vector transfected rMSCs, the green fluorescence expression in rMSCs were observed. The fluorescence expression was stronger along with increasing of MOI. When the MOI was up to 100, The fluorescence expression was strongest. And analysed with flow cytometer, the rate of transfection was on the increase along with increasing of MOI. The rate was 100% when the MOI was 100. The level of hBDNFmRNA and hBDNF expression was also stronger along with increasing of MOI. And the level was strongest from 3 days to 10 days when the MOI was 100. Conclusions rMSCs were successfully transfected by the recombinant adenovirus vector Ad-hBDNF-EGFP. hBDNF gene-modified rMSCs could express BDNF and hBDNFmRNA abundantly, and which laid a foundation of further study on gene therapy of SCI in rats.Part ThreeObjectives To transplant intravenously hBDNF gene-modified rMSCs to adult rats of spinal cord injury, then observe the viability , the changes of the gene expressions and the neurological morphological repairing and functional recovery after spinal cord injury in adult rats.Methods The rat spinal cord injury model was prepared according to the modified Allen method and the injury level was in the T10 segment of spinal cord. The injured rats were randomly divided into 4 groups: sham operated group, control group, Ad- EGFP-rMSCs transplantation group, hBDNF-rMSCs transplantation group. At three days after preparation of models, 1ml 0.1M phosphate buffered saline (PBS), or 1ml hBDNF-rMSCs suspension, or 1ml Ad-EGFP-rMSCs suspension was infused into tail vein respectively. The neurological functions of rats were evaluated using the Basso-Beattie-Bresnehan Score (BBB) and cortical somatosensory evoked potential (CSEP) at 24 hours after injury and 1, 3, 5 weeks post-infusion respectively. The viability and distributing of rMSCs in the spinal cord were observed by fluorescent microscope and the level of hBDNF expression in the injured spinal cord were analyzed with Western blotting. The expressions of GFAP, NF-200 andSynaptophysinⅠwere analyzed with immunohistochemistry. The histomorphological changes of the injured spinal cord were observed by electron microscope.Results After transplanting there was significant neurological function improvement in rats treated with hBDNF-rMSCs and Ad-EGFP-rMSCs compared with that of control group. The improvement of BBB and CSEP in hBDNF-rMSCs transplantation group was significantly greater than those in the other two groups. In Ad-EGFP-rMSCs and hBDNF-rMSCs groups, the grafted rMSCs survived at the site of injured spinal cord and the green fluorescence expression in rMSCs were observed. hBDNF strong expression was examined in hBDNF-rMSCs group by Western blotting. The GFAP, NF-200 and SynaptophysinⅠpositive cells were found at post-transplantation in each group. And the expression was the strongest in hBDNF-rMSCs group. The histomorphology of injured spinal cord was restored better in the Ad-EGFP-rMSCs and hBDNF-rMSCs transplanted group than that in the control group.Conclusions Transplanted hBDNF-rMSCs are able to survive and assemble at the injured area of spinal cord, and express hBDNF there.Intravenous administration of hBDNF-rMSCs promotes the restoration of injured spinal cord and improves neurological functions.