The Therapeutic Efficacy Of Lentiviral Vector Mediated Bdnf Gene-modified RMSCs In Rats With Cerebral Infarction

Part OneObjectives To obtain intact and errorless rat brain-derived neurotrophic factor (Bdnf) gene. Construct lentiviral vector carrying the Bdnf gene, and then infect the rat mesenchymal stem cells (rMSCs) to get Bdnf gene-modified rMSCs.Methods The cDNA encoding the CDS of the rat Bdnf gene was obtained from the hippocampus of the adult Fisher 344 rats by RT-PCR. The transfer plasmid of LV: PNL-BDNF-IRES2-EGFP was constructed, the three plasmids system of lentiviral vector: PNL-BDNF-IRES2-EGFP, HELPER and VSVG were co-transfected to 293T cells to produce Bdnf gene and the reporter gene enhanced green fluorescent protein (EGFP) gene co-expressing lentivirus. rMSCs were separated from the bone marrow of two-month -old F344 rats, cultured in vitro, and identified. rMSCs were infected by the lentivirus produced formerly and identified by fluorescent microscope, RT-PCR and immunofluorescent cytochemical staining.Results The result of sequencing showed that the cloned Bdnf gene is consistent with the sequence reported in the Genebank. The PNL-BDNF-IRES2-EGFP plasmid was identified to be correct. After three plasmids of lentiviral vector co-transfecting to the 293T cells, a great mount of green fluorescence in 293T cells was observed with the fluorescent microscope. The supernatant was collected and concentrated by ultracentrifugation and the titer of lentivirus measured was 6.7×107 TU/ml. After the...