ABSTRACT
BACKGROUND:Brain-derived neurotrophic factor (BDNF) has widespread effects on dopaminergic neurons, cholinergic neurons and other neurons, which can promote more differentiation of stem cells into neuron-like cells. Hypoxia inducible factor-1α(HIF1α) can improve tissue and cellviability in the ischemic environment and maintain the local microenvironment of hemangioblasts, which has a more far-ranging physiological role than genes. OBJECTIVE:By constructing double gene recombinant adenovirus vector with BDNF and three points mutant HIF1αto complete the transfection of adenovirus vector into rats mesenchymal stem cells and then study the promoting nerve regeneration and angiogenesis effect of these two genes to spinal cord injury in vitro in constant oxygen conditions. METHODS:(1)We finished the site-directed mutagenesis of 402, 564 and 803 amino acids in human HIF1αgene CDS region by PCR method and we finished recombination of mutation posterior HIF1αgene and BDNF into adenovirus pAdEasy-1 system. Packaging viral and titration determination were also finished. (2)Four kinds of virus fluids and blank group were selected for subsequent experiments. We observed transfection efficiency after transfection of virus into bone marrow mesenchymal stem cells and detected the expressions of BDNF and HIF1αmRNA and protein in transfection cells. (3) The protein expression of vascular endothelial growth factor, downstream formation vascular gene of HIF1α, in cells in al groups was detected by western blot assay. RESULTS AND CONCLUSION:(1)The site-directed mutagenesis of 402, 564 and 803 amino acids to alanine in coding sequence region was successful. The construction of four kinds of adenoviral recombinants was successful and identification of packaging was completed. (2)The level of BDNF gene mRNA and protein expression in experimental and positive control 1 groups was significantly higher than that in the other groups (P<0.05). The level of HIF1αmRNA and protein expression in experimental and positive control 2 groups was significantly higher than that of the other groups (P<0.05). The level of vascular endothelial growth factor protein was also increased in the experimental and positive control 2 groups, which was significantly different from other groups (P<0.05). These findings indicate that the BDNF and HIF1αproteins are largely and efficiently expressed in constant oxygen conditions after single vector double gene adenovirus system transfection into mesenchymal stem cells and high-efficiency expression of vascular endothelial growth factor protein is promoted so that this is a new direction for treatment of spinal cord injury by gene therapy combined with celltransplantation.