Studies On Fibroblasts Transfected With Vascular Endothelial Growth Factor Gene

Background: Closure of full-thickness skin defects is very importan to thetreatInent of patients with severe burns. As these patients with severe bams isconcemed, the donor site is far from enough, which results in many seriouscomplications, even death. The artificial skin can partly solve the problem.ReconstrUction of composite skin is the ultimate aim of skin tissueengineering. The composite skin is mainly composed of tIiree layers: epidermallayer, dermal analog layer and fibroblast layer. Many ldnds of artificial skinhave been used in clinical work, but they are not very satisfying. The artificialskin, or the dermal analog often has slow vascularization or poor take rate. It isthe key to improve the angiogenesis of the artificial skin fOr improving itspractical value.The use of biological agents to stimulate or augment angiogenesis is anexciting field of scientific research and there is optimism that it will become animportant therapeutic modality. The potential of exogenous agents to stimulatethe development of new blood vessels into ischaemic tissue has recently arousedconsiderable inierest.The result of angiogeneses is better oxygenation, provision of nutrients andremoval of waste products from tissues. To be able to augment this ntaalphysiological process with exogenous biological theraPies is an exciting conceptand would represent a major advance in medicine. The optimal agents, vectors,sites of administration and dosage strategies will take time to define. There isjustifiable optimism that theraPeutic angiogenesis could revolutionise thetTeatment of ischemia, bllt many questiol1s remain unanswered. More research isessential.Objcctivcsl. To amplify hVEGF,,, gene in vitro and constructing an eukaryoticexpression vector pcDNA3- hVEGF,,,;2. To transfer the recombinant vector into human fibroblast, and obtainingthe fibroblast cells transferred with pcDNA3- hVEGF,,,;3. To do some basic research about the transferred fibroblast cells.Material and Methodsl. Reversible transcription polymerase chain reaction/RT-PCR has beenaPplied to arnplify hVEGF,,, gene Which was then constructed to aneukaryotic expression vector pcDNA3;2. The recombinant vectoT, pcDNA3- hVEGF,,, was then trandferred intohuman fibrobIast celIs by FeGENE transfection regent. SeIected byGeneticin/G418, the fibroblast cells transferred with the recombinamvector were obtained,3. Some basic research were done on the trandferred cells f?To demonstrate VEGF mRNA expression in the transferred fibroblastcells by RT-PCR method (2) To demonstrate the secreted VEGF protein concentration in thesupernatant by Enzyme-Linked Immunosorbent Assay/ELISA method (D To draw the cell proliferation curve of the transferred cells by MTTmethod ?To determine the biological activity of the secreted protein byculturing the human umbilical vein endothelial cells/HUVEC with thesupernatant of the transferred fibroblast cells (5) To determine the biological activity of the secreted protein byVascular Permeability Assay. Conditioned medium obtained from thetransferred cells was tested in guinea pigs for its permeability activityin a Miles assayResults:l.The hVEGF165 gene has been amplified in vitro and an eukaryotic expression vector pcDNA3- hVEGF165 was successfully constructed;2. The fibroblast cells transferred with the recombinant vector were obtained;3. It was proved that the transferred cells demonstrated VEGF mRNA expression and the expression level was more than normal fibroblast celis. It was also proved that VEGF protein was secreted into the supernatant and the concentration was about lOpg/ml. Furthermore, the conditioned medium from these cells enhanced vascular permeability in vivo and improved the growth of the cultured HUVECs.Conclusions:The hVEGF165 gene was successfully cloned and transferred into fibroblast cells. The transferred cells demonstrated VEGF mRNA expression and protein secret...