Recombinant Adenovirus-mediated β-nerve Growth Factor Transfect Endothelial Progenitor Cells Of Rats

BackgroundSpinal cord injury is the most serious complications of spinal injury, occurring in falling injury and traffic accident injury and it has a high morbidity, showing severe limb dysfunction under the damage section. With the high-speed development of construction and transportation industry, there is an increasing incidence of spinal cord injury. It not only brings patients with serious physical and psychological harm, but also causes serious economic burden to society and family. However the current measures for the treatment of spinal cord injury were very limited and the repair and regeneration of neurons after injury were difficult in the treatment of spinal cord injury. With the rapid development of neurobiology, more and more researches focus on the field of stem cell transplantation for treatment of spinal cord injury. EPCs play an important role not only on the repair the damaged endothelial, but also in angiogenesis. Nerve growth factor has the function of nourish neurons, promoting the growth of neuron axons, and participating in the process of nerve regeneration after spinal cord injury repair. Adenovirus, as a carrier of most used in gene therapy, is widely used in the transfection of exogenous genes into cells. But the combination of them used to the treatment of spinal cord injury (SCI) is rarely reported. The endothelial progenitor cells (EPCs) from the bone marrow of rats were transfected with recombinant adenovirus carrying β-nerve growth factor (β-NGF) gene in our study and their effects were combined. β-nerve growth factor was continuously expressed in endothelial progenitor cells (EPCs) and it provides a new way of the treatment of spinal cord injury.ObjectiveTo explore the most suitable multiplicity of infection (MOI) which the endothelial progenitor cells (EPCs) from the bone marrow of rats were transfected with recombinant adenovirus carrying β-nerve growth factor (β-NGF) gene and observe the expression of β-NGF at genetic transcription and protein synthesis level after the transfection of recombinant adenovirus to EPCs and investigate the effect to microenvironmrnt of neuron after spinal cord injury.MethodsEPCs from the bone marrow of rats were isolated by density gradient centrifugation and cultured by the whole bone marrow adherent methods; Cell surface specific molecules such as CD34, CD 133 and VEGFR-2 were detected by immunofluorescence method. The most suitable MOI was identified by the transfection of recombinant adenovirus carrying β-NGF gene and green fluorescence protein (GFP) gene to EPCs with various MOI. The experiment group cells (group A) were transfected with recombinant adenovirus carrying β-NGF gene, the negative control group cells (group B) were transfected with vacant recombinant adenovirus and the blank control group cells (group B) were not treated. The expression of NGF was detected by real time-PCR (RT-PCR), western blot and enzyme-linked immuno sorbent assay (ELISA).ResultsEPCs from the bone marrow of rats were successfully isolated and cultured and CD34, CD 133 and VEGFR-2 were positive expression. The most suitable MOI we explored was 70, and the mRNA and protein expression of β-NGF were increased after adenovirus transfection.Conclusion1.EPCs were successfully transfected with recombinant adenovirus containing β-NGF, and the expression of β-NGF was increased at genetic transcription and protein synthesis level after the transfection of recombinant adenovirus carrying β-NGF to EPCs.2.The cells carrying β-NGF gene continually secreted an activeβP-NGF protein into extracellular matrix and the microenvironmrnt contributed to the repair and regenation of neuron after spinal cord injury.