The Research About The Effect Of Gene Gun-mediated Transfection With Human BFGF Gene On Deep Partial Thickness Burn Wound Healing

Background:The treatment of deep-partial thickness burn wound is a difficult problem in clinic. It usually takes 3-4 weeks to achieve wound healing through dress changing. The time of changing dressings is very long and the quality of woud healing is poor.The wound healing process of burn includes inflammatory reaction, cell proliferation and tissue molding, during which growth factors play a very important role. Basic fibroblast growth factor can promote proliferation and repairation of subcutaneous tissue, derma and epidermal cells effectively through several mechanisms. Applying basic fibroblast growth factor effectively in deep partial thickness burn treatment can accelerate wound healing process and improve wound healing quality.The modality to puffing and spreading growth factors onto wound could't exerts its best effectiveness, and suffers from problems with low bioavailability, stability and limited permeability in clinical work. Directly transferring gene into the wound is rational theoretically. Gene therapy should take two follow aspects into consideration: First the methods for gene transfer should do no harm or lillte damage to target cells or tissues. Second, the methods should reach enough transfection efficiency in target cells to produce therapeutic efficacy needed. The techniques for gene delivery frequently used nowadays have been faced some problems, mainly the low transfection efficiency, complicated gene transfer procedure and insufficient security, such as lipid and viral gene transfection.A microparticle bombardment system has been investigated for gene transfer in last decades. This system requries gold microparticles coated with plasmids loaded DNA in the presence of Ca²⁺, and then delivers gold microparticles into cultural cells, body surface or other exposed tissues by a special device called the gene gun using a high velocity supersonic flow of helium gas to accelerate the particles. DNA detach with gold microparticles inside the cells and then express their codogenic proteins. Little tissue damage was found because no cytotoxic effect was found of gold parti- cles in ex vivo or in vivo experiments, and particles have tiny size, about 1-3μm in diameter. Microparticle bombardment system is a convenient, high efficient and very safe system for gene transfer especially for cultural cells and body superficial parts for its characteristics. So this system has it distinct value for burn treatment. Methods:There were three experimental parts in this paper:Partâ… We extracted the former constructed human bFGF eukaryotic expression vector pCI-neo-bFGF. Then plasmid was incised with enzyme and characterizated.Partâ…¡Deep partial thickness burn woud model was created on dorsal skin of SD rat by contact for 12 second at 80℃using contrilling temperature unit (pathological section confirmed). The gene gun parameter (helium gas pressure) was optimized by using eukaryotic expression vector pEGFP which encoding green fluorescent protein.Partâ…¢We transfected SD rat deep partial thickness burn wound with pCI-neo-bFGF using gene gun, and negative control was set by transfecting empty eukaryotic expression vector pCI-neo. The time of wound healing was recorded when the burn wound was fully epithelized, the level of keratinocyte Collagenase-1 and hydroxyproline were determined using Elisa kit. At last we evaluated the effect of gene gun on deep partial thickness burn wound through comparing aforesaid indexes.Results:Partâ… I Sufficient plasmid pCI-neo-bFGF was gained after extraction, which concentration is 1μg/μl. The electrophoresis of extraction had shown that the strips are corresponding to bFGF genetic fragments and plasmid pCI-neo.Partâ…¡The pathological sections showed that deep partial thickness burn wound model was successfully created on dorsal skin of SD rat by contact for 12 second at 80℃. We proved that the gold microns can be delivered into the deep partial thickness burn wound of SD rat when helium gas pressure was set at 350psi (1 psi= 6890Pa) .Partâ…¢Afer transfecting deep partial thickness burn wound with bFGF gene using gene gun technology, we found that the time of wound healing was shorter, and the level of keratinocyte Collagenase-1 and hydroxyproline of burn wound were higher than negative control during wound healing progress.Conclusions:In this research 1μg/μl the former constructed human bFGF eukaryotic expression vector pCI-neo-bFGF we extracted. A deep partial thickness burn wound model of SD rat was successfully created using contrilling temperature unit. And optimized gene gun working parameter for deep partial thickness burn wound was gained. Finally we discovered that the wound healing process was accelerated by transfecting human bFGF gene into burn wound using gene gun, which appeared shorter wound healing period, higher level of keratinocyte Collagenase-1 and hydroxyproline during wound healing. All above approved that the modality of gene gun-mediated transfection with human bFGF gene was effective and feasible for deep partial thickness burn wound treatment.