siRNA介导低表达VEGF、SDF-1对血管内皮细胞增殖和凋亡的影响研究

Effects of siRNA-mediated low expression of vascular endothelial growth factor and SDF-1 on proliferation and apoptosis of vascular endothelial cells

目的研究小干扰RNA(siRNA)介导低表达基质细胞衍生因子-1(SDF-1)、血管内皮生长因子(VEGF)对血管内皮细胞增殖和凋亡的影响。方法采用体外实验培养人血管内皮细胞,细胞分别经siRNA介导下调表达SDF-1、VEGF处理,随机分为空白对照组(未进行任何转染)、阴性对照组(转染非特异性对照)、转染SDF-1-siRNA组、转染VEGF165-siRNA组。各组分别经相应处理。通过免疫印迹法测定细胞SDF-1和VEGF165蛋白的表达水平,用MTT实验测定人血管内皮细胞增殖情况,用流式细胞仪检测细胞凋亡情况。结果空白对照组与阴性对照组SDF-1和VEGF165蛋白表达水平的比较,无显著差异(P>0.05);相比空白对照组和阴性对照组,转染SDF-1-siRNA组细胞中SDF-1蛋白的表达水平显著下调(P<0.01),VEGF165蛋白表达并无显著差异(P>0.05);相比空白对照组和阴性对照组,转染VEGF165-siRNA组SDF-1和VEGF165蛋白表达水平均显著下调(P<0.01);转染VEGF165-siRNA组SDF-1蛋白表达水平显著高于转染SDF-1-siRNA组,VEGF165蛋白表达水平显著低于SDF-1-siRNA组(P<0.01)。空白对照组与阴性对照组细胞细胞凋亡率的比较,并无显著差异(P>0.05);转染SDF-1-siRNA组、转染VEGF165-siRNA组细胞增殖抑制率和细胞凋亡率均显著高于空白对照组和阴性对照组(P<0.01),转染VEGF165-siRNA组细胞增殖抑制率和细胞凋亡率均显著高于转染SDF-1-siRNA组(P<0.01)。结论SDF-1和VEGF165基因均可增强人血管内皮细胞增殖能力,阻滞细胞凋亡,从而参与糖尿病血管病变的病理过程,同时该病理过程中VEGF165具有调节SDF-1表达水平的作用。

Objective To investigate the effects of small interfering RNA(siRNA)mediated low expression of stromal cell derived factor-1(SDF-1)and vascular endothelial growth factor(VEGF)on proliferation and apoptosis of vascular endothelial cells.Methods Human vascular endothelial cells were cultured in vitro.The cells were treated with siRNA-mediated down-regulation of SDF-1 and VEGF.The cells were randomly divided into blank control group(without any transfection),negative control group(transfection non-specific control),SDF-1-siRNA transfection group and VEGF 165-siRNA transfection group.The expression levels of SDF-1 and VEGF165 protein were measured by immunoblotting,the proliferation of human vascular endothelial cells was measured by MTT,and the apoptosis of human vascular endothelial cells was detected by flow cytometry.Results There was no significant difference in the expression of SDF-1 and VEGF165 between the blank control group and the negative control group(P>0.05).Compared with the blank control group and the negative control group,the expression of SDF-1 protein in SDF-1-siRNA transfected cells was significantly down-regulated(P<0.01),and there was no significant difference in the expression of VEGF165 protein(P>0.05).Compared with the blank control group and the negative control group,the expression of SDF-1 and VEGF165 protein in the transfected group was significantly decreased(P<0.01);the expression of SDF-1 protein in the transfected group was significantly higher than that in the transfected group,and the expression of VEGF-165 protein was significantly lower than that in the SDF-1-siRNA group(P<0.01).There was no significant difference in the cell apoptosis rate between the blank control group and the negative control group(P>0.05).The cell proliferation inhibition rate and apoptosis rate in SDF-1-siRNA transfection group and VEGF165-siRNA transfection group were significantly higher than those in the blank control group and the negative control group(P<0.01).The cell proliferation inhibition rate and cell apoptosis rate in the transfected group were significantly higher than those in the blank control group and the negative control group(P<0.01).The apoptotic rate was significantly higher in SDF-1-siRNA transfection group than that in SDF-1-siRNA transfection group(P<0.01).Conclusion Both SDF-1 and VEGF165 genes can enhance the proliferation of human vascular endothelial cells and block cell apoptosis,thus participating in the pathological process of diabetic angiopathy.At the same time,the expression level of SDF-1 can be regulated by VEGF-165 in this pathological process.