论文部分内容阅读
目的：探讨SIRT3基因对氧化型低密度脂蛋白（ ox-LDL）刺激的人主动脉内皮细胞（ HAEC）炎性因子细胞间黏附分子-1（ICAM-1）、E-选择素（E-selectin）表达的影响。方法将生长状态良好的细胞随机分为空白对照组、转染试剂对照组、阴性对照组及SIRT3 siRNA（1、2、3、4）组。筛选出最佳序列后，细胞分为空白对照组、siRNA组、（20、30、40μg/mL）ox-LDL组、siRNA＋（20、30、40μg/mL）ox-LDL组。转染24 h、ox-LDL刺激6 h。 qRT-PCR检测SIRT3 mRNA水平，Western blotting检测SIRT3蛋白表达，流式细胞术检测E-selectin、ICAM-1抗体的平均荧光强度值（MFI）。结果 SIRT3 siRNA2序列在mRNA水平的干扰效率为88．62％，蛋白水平为80．37％，干扰效果明显。与空白对照组相比，ox-LDL各组的SIRT3 mRNA相对表达量差异有统计学意义（P均＜0．05）；随ox-LDL浓度增加，SIRT3 mRNA相对表达量逐渐降低（ P均＜0．05）。与siRNA组相比，siRNA＋ox-LDL各组ICAM-1、E-selectin的MFI差异有统计学意义（P均＜0．05）；随ox-LDL浓度增加，MFI逐渐升高（P均＜0．05）。与空白对照组相比，siRNA组ICAM-1、E-selectin的MFI无统计学差异（P均＞0．05）。结论 SIRT3基因与ox-LDL刺激的人主动脉内皮细胞ICAM-1、E-selectin表达升高相关，可能参与并调控动脉内皮细胞损伤的炎症反应。“,”Objective To investigate the effect of SIRT3 gene on the expressions of inflammatory factors intercellular cell adhesion molecule-1 ( ICAM-1) and E-selectin in human aortic endothelial cell ( HAEC) stimulated by oxidized-low density lipoprotein( ox-LDL) .Methods The cells were randomly divided into normal control group, transfection reagent control group, negative control group, and SIRT3 siRNA (1, 2, 3 and 4) groups.When the most effectiveinterference sequence was selected out, the cells were divided into normal control group, siRNA group, (20, 30 and 40μg/mL) ox-LDL groups and siRNA+(20, 30 and 40μg/mL) ox-LDL groups.The cells were transfected for 24 h and stimulated by ox-LDL for 6 h.SIRT3 mRNA level was detected by qRT-PCR and its protein level was analyzed by Western blotting.Mean fluorescence intensities ( MFIs) of ICAM-1 and E-selectin antibody were detected by flow cytometry.Results SIRT3siRNA2 was the most effective interference sequence.The interference efficiency of SIRT3siRNA2 was 88.62%in mRNA level and 80.37%in protein level.Expressions of SIRT3 mRNA had significant differences in ox-LDL groups (all P<0.05) compared with that in normal control group;with the increase of ox-LDL concentration, the expression of SIRT3 mRNA gradually declined (all P<0.05).MFI of ICAM-1 and E-selectin had statistical differences in siRNA+ox-LDL groups compared with that in siRNA group ( all P<0.05);with the increase of ox-LDL concentration, the MFI was enhanced obviously (all P0.05).Conclusion The SIRT3 gene has association with the expressive increments of E-selectin and ICAM-1 in HAEC stimulated by ox-LDL, and may regulate the inflammatory reaction in artery endothelial cell injury process.