Studies On The Molecular Signal Pathway In The Spinal Cord Neuroplasticity Following BDNF Transgenic Fibroblast Transplantion

Objective To establish reliable spinal cord transected injury models in adult rats, construct the fibroblasts (FB) modified with BDNF gene, then explores the effect of BDNF gene-modified fibroblasts engrafted into the injured spinal cord in rats on functional recovery and the regulation of molecular signal pathway.Methods After T8 vertebral lamina and adjacent ligamenta flava were removed from adult Sprague-Dawley (SD) rats, spinal cord was fully transected by using eye scissors in order to duplicate spinal cord transsected-injury(SCT) model, which is dependent on the changes in somato-sensory evoked potential (SEP), motor evoked potentials (MEP), pathologic and Basso, Beattie and Bresnahan (BBB) scores examinations.This was followed to construct brain-derived neurotrophic factor (BDNF) gene clone vector after BDNF was amplified by RT-PCR from rat brain tissue. By using gene recombination technique, BDNF coding sequence was inserted into eukaryotic expression vector pcDNA3. The recombinant plasmid was verified with restriction enzyme digestion and DNA sequencing. Fibroblasts were transfected with the recombinant vector using LipofectamineTM 2000 transfection reagent. The expression of BDNF was analyzed by immunocytochemistery and western blot, and biologic activity of BDNF was determined by using PC12 cells in vitro.To study the behavior of rats, the rats were devided into SCT group, FB transplantation group (FB group), BDNF-FB transplantation group (BDNF-FB group). All aminals were transected at the T10 segment of spinal cord. At 7 days after preparation of models,5μl Sodium Chloride was infused into the spinal cord of SCI group,5μl FB and BDNF-FB labelled with Hoechst33342 was transplanted into the spinal cord of FB group and BDNF-FB group, respectively. At 21 days after preparation of models, spinal cords and gastrocnemius muscles were taken out from each group, then performed RT-PCR detection for cyclin D1, Cyclin E, CDK4, Bad, bcl-xl, CREB, p90rsk and Stat3 expression. The motor function of the rest rats were evaluated using the BBB Score at 24 hours before and after injury and 1,2,3,4,5 weeks after injury. After infusion respectively, the localiztion of FB and BDNF-FB in the spinal cord was observed by fluorescent microscope and the level of BDNF expression in the injured spinal cord were analyzed with with immunohistochemistry.ResultsAfter SCI, BBB scores of SCT group in days 1,3,5,7 were respectively less than that in control group. The differences were significant(P<0.01). The SEP and MEP in SCT group were not evoked out; while it showed the normal waveshapes in control group. After perfused with paraformaldehyd, the spinal cords of control group were intact; that of expermiental group were fully transected.RT-PCR showed that BDNF product is 749bp specific segment. By restriction enzyme digestion, the recombinant plasmid consisted to 749bp and 5.4kb fragments. The DNA sequence of the 749bp fragment was identical with rat BDNF cDNA in GeneBank. The immunocytochemistery and western blot showed the BDNF was expressed successfully in fibroblasts. And the culture medium of BDNF-gene modified fibroblasts could promote the PC12 cells growth in vitro.The improvement of BBB Scores in BDNF-FB group was significantly greater than those in the other two groups (P<0.01). In FB and BDNF-FB groups, the grafted cells survived at the site of injured spinal cord and the blue fluorescence expression in FBs and BDNF-FBs were observed. BDNF strong expression was examined in BDNF-FB group by immunohistochemistry. The expression of cdk4 and CyclinDl in spinal cord of BDNF-FB group decreased significantly than those in the other two groups (P<0.05), while the expression of CyclinD1 in gastrocnemius muscles of BDNF-FB group increased significantly, compared with that of the other two groups (P<0.05). The expression of CREB in gastrocnemius muscles of BDNF-FB group decreased significantly than those in the other two groups (P<0.05)Conclusion SCT model is established successfully, by evaluation from histological, electrophysiological and behavior. The recombinant plasmid pcDNA3/BDNF was constructed successfully, which will provide the foundation for the further research. Transplanted BDNF-FBs could survive at the injured area of spinal cord, and express BDNF protein. Moreover, transplantion of BDNF-FBs promotes the restoration of injured spinal cord and improves motor functions. The mechanism may be related with the molecular signal pathway about CyclinD1, cdk4 and CREB in spinal cord and muscle.