摘要
目的 观察质粒DNA在大鼠术后肠黏连模型中的表达效率及肝细胞生长因子(HGF)基因转移对术后肠黏连的预防作用.方法 以pcDNA3-HGF转染CHO细胞24 h后收集培养上清,用ELISA测定培养上清中HGF的浓度;取培养的大鼠腹膜间皮细胞作划痕试验,观察表达HGF的培养上清对间皮细胞迁移的影响.利用无菌干纱布擦伤盲肠的方法建立Wistar大鼠术后肠黏连模型,将模型大鼠随机分为5组,每组10只.其中4组分别于创面涂布pcDNA3-HGF 10、50、100和200 μg,1组于创面涂布绿色荧光蛋白(GFP)表达质粒pcDNA3-GFP 200 μg作为对照.术后第2、14天分别解剖pcDNA3-GFP 200 μg组大鼠各2只取腹膜组织,荧光显微镜下观察GFP的表达;术后第14天解剖各组动物,观察肠黏连的发生情况.结果 pcDNA3-HGF质粒转染CHO细胞后24 h培养上清的HGF浓度达40 ng/ml,该上清能够促进大鼠腹膜间皮细胞的迁移.pcDNA3-GFP 200 μg组大鼠术后第2天腹膜组织中观察到GFP的表达,并持续到术后第14天.术后第14天各组大鼠中均有发生不同程度肠黏连者,pcDNA3-HGF 10、50、100和200 μg组和pcDNA3-GFP 200 μg组大鼠肠黏连发生率分别为9/10、7/10、6/10、4/10和9/10,其中重度(3～4度)肠黏连发生率分别为5/10、5/10、2/10、3/10、7/10,pcDNA3-HGF对术后肠黏连的预防效果呈一定的量效关系.结论 质粒DNA能有效转染损伤腹膜并介导外源基因表达;质粒载体介导HGF基因转移是预防术后肠黏连的有效手段.
Objective To investigate the transfection efficiency of plasmid DNA,and preventive effect of hepatocyte growth factor(HGF) gene transfer on postoperative intestinal adhesion in rats. Methods The concentration of HGF in the supernate of pcDNA3-HGF transfected CHO cells was determined by ELISA. The effect of HGF on mesothelial cell migration was investigated by scratch wound motility assay. A rat model of cecal adhesion was established to evaluate the preventive effect of plasmid-mediated HGF gene transfer on postoperative intestinal adhesion. The animals were allocated randomly to the treatment(n = 40) and control(n = 10) groups. In the treatment group,10,50,100,or 200 μg pcDNA3-HGF were painted onto the surfaces of the injured cecum,respectively(n = 10 for each subgroup). In the control group,pcDNA3-GFP 200 μg was applied. All the animals were sacrificed on day 2 and day 14 postoperatively. The expression of GFP was detected to determine the transfection efficiency of plasmid DNA in the rats. Results The concentration of HGF in the supernate of pcDNA3-HGF transfected CHO cells was 40 ng/ml. The supernate stimulated the migration of mesothelial cells. In the control group,GFP was expressed on day 2,and persisted for at least 2 weeks. The incidence of cecal adhesion was 9/10,7/10,6/10,and 4/10 in 10,50,10,and 200 μg pcDNA3-HGF subgroups,and was 9/10 in the control group;while the incidence of substantial adhesion(grade 3 to grade 4) was 5/10,5/10,2/10,3/10,and 7/10,respectively. Plasmid DNA could effectively transfect the damaged mesothelium in a rat model. Local application of plasmid carrying HGF gene reduced the rate of severe postoperative intestinal adhesion. Conclusion Plasmid-mediated HGF gene transfer may be a novel strategy for preventing postoperative intestinal adhesion.
出处
《中国医药生物技术》
CSCD
2007年第3期189-193,共5页
Chinese Medicinal Biotechnology
关键词
肝细胞生长因子
黏连
手术后并发症
质粒
间皮细胞
Hepatocyte growth factor
Adhesion
Postoperative complications
Plasmid
Mesothelial cells
