Resumo

After brain injury, endogenous neural stem cells (eNSCs) resident in the subventricular zone are recruited by chemokines liberated at the injury site, and one of the most potent chemokine that attract eNSCs is CXCL12/SDF-1. Although a great number of eNSCs is attracted, not enough cells arrive and survive at the injury site leading to poor functional recovery. We aim to develop strategies to increase the number of eNSCs attracted to injury sites as well as to improve survival of those cells in order to increase the chances of tissue regeneration and, consequently, functional recovery. Using a model of brain trauma applied to the motor cortex of mice we observed that synthetic peptides analogous to the N-terminal of CXCL12/SDF-1 injected at the injury site increased the amount of eNSCs attracted. Because the synthetic peptides are very labile when applied to the injured tissue we decided to try a combined cell and gene therapy approach. In order to do that, we decided to clone CXCL12/SDF-1 complete gene as well as parts of the N-terminal (17-21aa) into a retrovirus and transduce mesenchymal stem cells that will be transplanted at the injury site and produce the chemokine or chemokine peptides into the injury. cDNA was obtained by reverse transcription of RNA extracted from bone marrow obtained from 2 months old female C57BL/6 mice. Initially, for CXCL12/SDF-1 complete sequence amplification, a pair of primers was designed with sites for EcoRI and KpnI restriction enzymes in order to insert the cloned gene into the vector of choice, pEGFP-N3, that has resistance markers to kanamycin and neomycin. The amplified product corresponding to CXCL12/SDF-1 complete gene was sequenced and inserted into the vector. The next step is to transduce mesenchymal stem cells and transplant them into the injury sites. Support: FAPESP, CNPq.