基因枪转人碱性成纤维细胞生长因子基因促进深Ⅱ度烧伤创面的愈合

背景:大量的体内和体外实验已证实,碱性成纤维细胞生长因子对不同组织和细胞具有广泛作用,可加快创面愈合进程.目的:观察基因枪转人碱性成纤维细胞生长因子基因促进深Ⅱ度烧伤创面愈合的效果和可行性.设计、时间及地点:完全随机设计,观察实验,于2007-12/2008-10在解放军第二军医大学长海医院全军烧伤中心实验室完成.材料:SD清洁级大鼠,体质量200-250 g,雌雄不拘.方法:将天然人碱性成纤维细胞生长因子基因重组优化,以pCI-neo为载体构建重组人碱性成纤维细胞生长因子基因高效真核表达载体pCI-neo-bFGF,并转染人胚肾细胞293T细胞,转染后以dot blot和western blot检测碱性成纤维细胞生长因子的表达.利用基因枪技术对SD大鼠深Ⅱ度烧伤创面模型进行转基因,以转染pCI-neo-bFGF为实验组,以转染空载体pCI-neo为对照组.主要观察指标:记录创面愈合时间,在转基因后24 h,48 h,96 h,7d,10d和14d测定创面组织羟脯氨酸和胶原酶Ⅰ水平,评价创面愈合情况.结果:重组构建的pCI-neo-bFGF经转染人胚肾细胞293T细胞,dot blot和western blot 检测结果显示,构建的pCI-neo-bFGF表达载体可表达人碱性成纤维细胞生长因子,荧光显微镜下合成基因的表达水平明显高于天然基因表达;基因枪转基因实验结果显示,实验组创面愈合时间为(13.00±1.31)d,对照组为(14.75±1.28)d,两组相比较差异有显著性意义(P＜0.05):两组羟脯氨酸及胶原酶Ⅰ水平均于基因枪转染后48h即伤后第5天达到高峰,随后逐渐下降至一定水平后维持,实验组各时间点羟脯氨酸水平均高于对照组(P＜0.05,P＜0.01);实验组转基因后48 h和96 h胶原酶Ⅰ水平明显高于对照组(P＜0.01).结论:基因枪转人碱性成纤维细胞生长因子基因可以缩短创面愈合时间.增加创面愈合期间组织羟脯氪酸和胶原酶Ⅰ水平,加快深Ⅱ度烧伤创面愈合进程.

Gene gun-delivered human basic fibroblast growth factor gene facilitates the healing of deep partial thickness burn wounds

BACKGROUND: A large amount of in vivo and in vitro experiments have confirmed that, basic fibroblast growth factor (bFGF) has been widely utilized in various tissues and cells, it can facilitate the wound healing.OBJECTIVE: To observe the efficacy and feasibility of gene gun-mediate delivery of human bFGF on the healing of deep partial thickness bum wounds.DESIGN, TIME AND SETTING: Randomized design,an observational trial was performed at the Military Central Laboratory of Changhai Hospital in the Second Military Medical University of Chinese PLA between December 2007 and October 2008.MATERIALS: SD rats of clean grade, weighing 200-250 g, irrespective of genders, ware involved in this study.METHODS: Natural human bFGF gene was recombined and optimized, then eukaryotic expression vector pCI-neo-bFGF was constructed taking pCI-neo as a vector, and transfeoted with human embryonic kidney cells 293 T cells. Dot blot and Western blot methods were utilized to determine the bFGF expression. Rat model of deep partial thickness burn wounds was processed into transgene process using gene gun technique, pCI-neo-bFGF-transfected ones served as experiment group while pCI-neo-transfected ones served as controls.MAIN OUTCOME MEASURES:Wound healing time was recorded and the efficacy was evaluated. The contents of hydroxyproline and collaganase Ⅰ in burn wound tissues were determined at 24 hours, 48 hours, 96 hours, 7 days, 10 days and 14 days following transgene process.RESULTS: the recombinant pCI-neo-bFGF was transfected with human embryonic kidney 293T cells. Dot blot and Western blot analysis have showed that, the constructed pCI-neo-bFGF expression vector could express human bFGF, and the expression of synthesized gene was remarkably higher than that of natural gene under fluorescence microscope; gene gun-mediated transgene experiment have showed that, the wound healing time was (13.00+1.31) days in the experiment group and (14.75±1.28) days in the control group, with significant differences (P＜0.05). The contents of hydroxyproline and collagenase Ⅰ reached a peak at 5 days after the injury, that is 48 hours after transfection, and then gradually decreased and maintained at a certain level. The experiment group had higher hydroxyproline levels compared with control group at different time points (P＜0.05, P＜0.01); the collagenase Ⅰ in the experiment group was notably higher than that in the control group at 48 hours and 96 hours after transfection (P＜0.01).CONCLUSION: Gene gun-mediated delivery of human bFGF can short the time of wound healing, increase the contents of hydroxyproline and collagenase Ⅰ during the healing period, accelerate the healing of deep partial thickness burn wounds.