携带SPK1基因的质粒预防术后肠粘连的实验研究

Preventive effect of plasmid-mediated sphingosine kinase1 gene transfer on intestinal adhesion after operation

目的观察质粒pcDNA3-SPK1在大鼠术后肠粘连模型中的表达效率及鞘氨醇激酶(SPK1)基因转移对术后肠粘连的预防作用。方法大鼠腹膜间皮细胞分离培养,pcDNA3-SPK1质粒转染大鼠腹膜间皮细胞,Westrn bolt检测SPK1蛋白的表达和γ-P32掺入法检测SPK酶活性;利用绿色荧光蛋白(GFP)为标志基因,评价质粒载体在手术损伤腹膜中的基因转移效率;利用无菌干纱布擦伤盲肠,建立大鼠术后肠粘连模型,评价SPK1基因转移对术后肠粘连的预防作用。结果 pcDNA3-SPK1质粒能有效转染损伤腹膜并介导SPK1基因表达;在大鼠术后肠粘连模型中,pcDNA3-SPK1基因转移能使术后重度肠粘连(3-4°)的发生率从80%降至30%。结论质粒pcDNA3-SPK1可有效预防术后肠粘连的发生。

Objective To observe the expression of plasmid sphingosine kinase1（SPK1） in a rat model of intestinal adhesion and the protective effect of SPK1 gene transfer on intestinal adhesion in rats after operation.Methods Mesothelial cells isolated from rats were cultured and transfected with pcDNA3-SPK1.Expression of SPK1 protein was detected by Western blot and activity of SPK1 was assayed by ATP incorporation.Transfer efficiency of plasmid vector in mesothelial cells was evaluated using green fluorescent protein（GFP） as a marker.A rat model of cecum brush burn-induced adhesion was established to evaluate the preventive effect of plasmid-mediated SPK1 gene transfer on intestinal adhesion after operation.Results Plasmid pcDNA3-SPK1 effectively transfected the damaged mesothelium and mediated the SPK1 gene expression.Plasmid pcDNA3-SPK1 gene transfer decreased the intestinal adhesion from 80% to 30% in the rat model of intestinal adhesion after operation.Conclusion Plasmid SPK1 can effectively prevent intestinal adhesion in rats after operation.