title: BDNF and NT-3 expression by using glucocorticoid-induced bicistronic expression vector pGC-BDNF-IRES-NT3 protects apoptotic cells in a cellular injury model.
abstract: Spinal cord injury (SCI) is a severe traumatic disease in the central nervous system with high incidence and high morbidity. Recent study demonstrated that cell transplantation therapy may improve local microenvironment of the injury site and promote nerve regeneration to restore spinal cord functions. In this study, we constructed a glucocorticoid-induced bicistronic eukaryotic expression vector pGC-BDNF-IRES-NT3 by using molecular cloning techniques and examined the protective effect of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) expressed by this vector in a rat spinal cord injury (SCI) model. We first connected glucocorticoid response element (GRE) to cytomegalovirus (CMV) promoter and then the GRE-CMV gene was inserted into pEGFP-1 vector to construct the eukaryotic expression vector pGC-EGFP. Western blot analysis was used to confirm the expression of EGFP by transfecting this vector in RN-DSC cells. The IRES was used to connect BDNF gene and NT-3 gene and replaced the EGFP gene in pGC-EGFP plasmid to form the bicistronic expression vector-pGC-BDNF-IRES-NT3. After RN-DSC cells were transfected with the plasmid and treated with glucocorticoid, BDNF and NT-3 expression in the culture medium were measured by ELISA method. Finally, we found that combination therapy with the transfection of this vector and glucocorticoid had an anti-apoptotic effect in a cellular SCI model of RN-DSC cells. Therefore, the co-expression of BDNF and NT-3 by using this vector rescued the injured cells. This provided useful information for the gene-modification cell transplantation combined with glucocorticoid for the treatment of SCI.
authors.affiliation: Department of Orthopedic Surgery, Clinical Medical College of Yangzhou, University of Yangzhou, Yangzhou City, Jiangsu province, 225001, China.
authors.forename: Yongxiang
authors.initials: Y
authors.lastname: Wang
authors.forename: Jiaxiang
authors.initials: J
authors.lastname: Gu
authors.forename: Jingcheng
authors.initials: J
authors.lastname: Wang
authors.forename: Xingmin
authors.initials: X
authors.lastname: Feng
authors.forename: Yuping
authors.initials: Y
authors.lastname: Tao
authors.forename: Baichuan
authors.initials: B
authors.lastname: Jiang
authors.forename: Jinshan
authors.initials: J
authors.lastname: He
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wang
authors.forename: Jiandong
authors.initials: J
authors.lastname: Yang
authors.forename: Shenfei
authors.initials: S
authors.lastname: Zhang
authors.forename: Jun
authors.initials: J
authors.lastname: Cai
authors.forename: Yu
authors.initials: Y
authors.lastname: Sun
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D019208:Brain-Derived Neurotrophic Factor / Q000096:biosynthesis*; D002460:Cell Line; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics*; D005938:Glucocorticoids / Q000494:pharmacology*; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008856:Microscopy, Fluorescence; D050506:Mutant Chimeric Proteins / Q000235:genetics; D009474:Neurons / Q000502:physiology*; D020933:Neurotrophin 3 / Q000096:biosynthesis*; D010957:Plasmids / Q000235:genetics; D051381:Rats; D020218:Response Elements / Q000235:genetics; D013116:Spinal Cord / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014162:Transfection
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D017931:DNA Primers; D005938:Glucocorticoids; D007202:Indicators and Reagents; D050506:Mutant Chimeric Proteins; D020933:Neurotrophin 3; D049452:Green Fluorescent Proteins