| Background:Chronic wounds is a common surgical disease, There are complex and multiple causes of the pathological changes. Due to poor tissue regeneration, wound healing difficulties. The present treatment of chronic wounds in addition to surgical removal of necrotic tissue, they are all relying on external drugs or dressings carried out,basing on improving the body state to control the primary disease. To stimulate angiogenesis, improve blood circulation, enhance tissue regeneration capacity and promote wound healing of chronic wounds healing to become a current research focus because ischemia plays an important role of the formation and occurrence of chronic wounds. Recently,bioactive growth factors such as TGF-beta,bFGF and neurotransmitters factor CGRP(calcintonin-gene related peptide) as well as asoactive intestinal polypeptide are applied eye-catching. BFGF is an important angiogenesis-stimulating factor and strongest mitogenic hormone known at present. Not only can It promote a variety of cell division and proliferation, chemotaxis and accelerate angiogenesis, but also play an important role in tissue repair, wound healing process. BFGF protein factor be directly effect on the wounds, its therapeutic effect is limited, and susceptible to a variety of enzymatic degradation within the wound. Continuing the direct administration of bFGF treatment cost is too high. There are hot researches through vector-mediated gene transfer contingent method to used to wounds, because bFGF protein factor can continue to improve blood circulation, enhance tissue regeneration capacity, promote wound healing. But the disadvantages of currently growth factor gene therapies are run transfer inefficient, vector targeting specific poor, and gene expression time shorter, affecting the efficacy. Therefore, we adopted the experimental design to build out a new type of chimeric recombinant adenovirus, to Increase its contingent transfer efficiency, improve targeting and enhanced gene expression of bFGF. We apply the chimeric recombinant adenovirus-mediated bFGF to act on the chronic ischemic rabbits ear wounds.Objective:To apply ischemic chronic wounds of rabbit ear model and use transformed with high efficiency through the ability to infect and targeted expression of adenovirus vector, to mediated human basic fibroblast growth factor (bFGF) expression. To study whether it can promote angiogenesis, enhance tissue regeneration, promote healing of chronic wounds and the protective effect. Final, respectively compare and evaluate the different effects and influences between the chimeric recombinant adenovirus and additional adenovirus-mediated bFGF, and exogenous bFGF on wound healing.Contents and Methods:1. Construction, identification and amplification of recombinant chimeric adenovirus Ad5F35-ET-bFGF. EDN1 promoter was amplified by PCR then inserted into pre-contructed shuttle vector pDC316-bFGF adenovirus to get pDC316ET-bFGF, then the shuttle plasmid pDC316ET-bFGF and Ad5/F35 chimeric adenovirus backbone plasmid pPE3-F35 was co-transfected into packaging cells to get the correct recombinant clone, and the virus plaques were picked and the right one was verified by both PCR method and gene sequencing, and then right viruses were amplified via repeated infection of 293 cells, a certain titer of adenovirus, by cesium chloride density gradient centrifugation, purified virus, the last round amplification viral titer was calculated through TCID50.2. Eestablished the model of chronic ischemic wound in rabbits ears and detected the expression of recombinant adenovirus in the wound. Made a 1cm diameter circular skin defect area on the dorsal of ear in New Zealand White Rabbit, until the cartilage exposed. To evaluate the reliability and feasibility of the model and detect the adenovirus expression in wound through general visual observation, HE stain, wound healing rate calculation, skin temperature measurement, blood gas analysis and fluorescence immunoassay methods.3. Chimeric recombinant adenovirus-mediated bFGF gene therapy on rabbit ears in the chronic wounds. Ad5F35-ET-bFGF group, Ad5-bFGF group, exogenous bFGF group, PBS group and blank control group were respectively divided into five groups in This experiment. On No. 1,3,5,7,9,14d postoperation, used micro-syringe multi-point(10 points) to inject subcutaneously Experimental reagents on each edge of rabbit ears wound. Each Ad5F35-ET-bFGF treatment wounds was injected into 108 pfu/ml recombinant chimeric adenovirus of Ad5F35-ET-bFGF, viruses were diluted to total 1ml with phosphate buffered saline (PBS),0.1ml per point. Ad5-bFGF treatment group wounds were injected 108 pfu/ml of Ad5-bFGF 1ml with the same method; exogenous bFGF group wounds were injected 108 pfu/ml of recombinant bovine bFGF lml with the same method; PBS control group wounds were injected equivalent PBS lml; Blank control group was not to intervene. Detected the expression of bFGF in the wound By gross observation, HE staining and immunohistochemistry, PCR, Western methods. Evaluated different effects and impaction between recombinant chimeric adenovirus-mediated bFGF and traditions adenovirus-mediated bFGF and exogenous bFGF on wound healing.Results:1. Constrated and established END1 promoter carrying with bFGF gene with a high degree of infection and specific targeted expression of the chimeric adenovirus-based value-added Ad5F35-ET-bFGF by homologous recombination technology. The viral titer was determined of 5.56×1010 pfu/ml.2. Established ischemic and chronic wounds model in rabbit ear. The ischemic and chronic wounds model confirmed a reliable, stable and highly feasible by using methods with gross observation, HE stain and wound healing rate calculation. Fluorescence immunoassay showed adenovirus transfect into the wound tissues successfully. It was positive expression and continued delivered at least 2 weeks in the cell.3. HE staining and immunohistochemical staining both showed Ad5F35-ET-bFGF persistent high expression in the chronic wound tissues. Wsetern Blot semi-quantitative detection certified bFGF protein over-expression in the wound for at least 2 weeks. Fluorescence quantitative PCR detection proved that the wound's mRNA level of bFGF was significantly increased after transfection. All results have shown that Ad5F35-ET-bFGF treatment of chronic wounds was superior to any other experimental group and control group.Conclusion:Our experiment established the ischemia and chronic wounds of rabbits ears model, based on building with high infection efficiency and specificity of targeted expression of the chimeric recombinant adenovirus Ad5F35-ET, mediated bFGF gene therapy for ischemia and chronic wounds. The study confirmed that its treatment of chronic wounds with good results. There were some of the research methods improved, and a few of features and innovative technologies produced by Experiment. It may represent the future development direction of bFGF gene therapy.
|
PDF Full Text Request