【摘要】 目的大量研究表明,内皮细胞的凋亡参与了动脉粥样硬化的发生与发展。另外有研究表明,细胞信号转导抑制因子-1(SOCS1)的表达下调可促进某些细胞的凋亡。然而,SOCS1表达下调是否与内皮细胞的凋亡有关,目前未见相关报道。为此,本研究利用RNA干扰沉默SOCS1的表达,然后观察内皮细胞凋亡的变化,探讨SOCS1与动脉粥样硬化之间的关系。方法 (1)培养人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cel,HUVEC),利用RT-PCR和Western Blotting检测SOCS1在HUVEC中的表达。(2)设计并化学合成4对靶向SOCS1的siRNA:siRNA-1、siRNA-2、siRNA-3、siRNA-4。(3)将带有荧光标记的阴性对照siRNA配制成25、50、75、100 nmol/L的4组,利用脂质体转染细胞,并在荧光显微镜下观察转染效率,得出有最佳转染效率的浓度。(4)HUVEC根据不同的处理因素分成8组:siRNA-1、siRNA-2、siRNA-3、siRNA-4和阳性对照组、阴性对照组、转染试剂组、空白对照组。将均为有最佳转染效率浓度的各组siRNA转染细胞,48小时后收获细胞。利用RT-PCR和Western Blotting,筛选出1对对SOCS1沉默效率最高的siRNA。(5)将筛选出来的沉默效率最高的siRNA和阴性对照siRNA以有最佳转染效率的浓度转染细胞,转染24小时后分为4组:①阴性对照siRNA;②阴性对照siRNA+缺氧20小时/复氧4小时;③SOCS1 siRNA;④SOCS1 siRNA+缺氧20小时/复氧4小时。最后,利用Western Blot检测Caspase3和Bax的表达,利用流式细胞仪检测凋亡。结果 (1)siRNA在50 nmol/L时有较高的转染效率。(2)4对siRNA干扰后,SOCS1的表达水平与四个对照组相比明显下调(P<0.05),其中siRNA-3的沉默效率最高(P<0.05)。(3)对HUVEC进行RNA干扰和缺氧复氧处理后,SOCS1 siRNA+缺氧复氧组的Caspase3、Bax蛋白表达水平与其它3组相比明显升高(P<0.05),阴性对照siRNA+缺氧复氧组的Caspase3、Bax蛋白表达水平与阴性对照siRNA组相比明显升高(P<0.05),阴性对照siRNA+缺氧复氧组的Caspase3蛋白表达水平与SOCS1 siRNA组相比明显升高(P<0.05),而阴性对照siR-NA组与SOCS1 siRNA组之间的Caspase3蛋白表达水平差别无统计学意义(P>0.05),SOCS1 siRNA组的Bax蛋白表达水平与阴性对照siRNA组相比明显升高(P<0.05)。(4).对HUVEC进行RNA干扰和缺氧复氧处理后,SOCS1siRNA+缺氧复氧组的凋亡率与其它3组相比明显升高(P<0.05)。阴性对照siRNA+缺氧复氧组的凋亡率与阴性对照siRNA组、SOCS1 siRNA组相比明显升高(P<0.05)。而阴性对照siRNA组与SOCS1 siRNA组之间的凋亡水平差别无统计学意义(P>0.05)。结论 (1)RNA干扰能有效抑制SOCS1在内皮细胞中的表达。(2)沉默SOCS1表达可加剧缺氧复氧导致的内皮细胞凋亡。更多还原

【Abstract】 Objectives It is well known that apoptosis of endothelial cell(EC) involves the development of atherosclerosis. Down-regulation of SOCS1(Suppressor of cytokine signaling-1) could induce apoptosis of a variety of cells.However, it has not been reported whether there is any similar phenomenon in EC.Here,we investigated the effect of SOCS1 silencing on Human Umbilical Vein Endothelial Cell(HU-VEC) apoptosis induced by hypoxia/reoxygenation and its association with atherosclerosis.Methods(1) SOCS1 expression in Human Umbilical Vein Endothelial Cell(HUVEC) was determined by RT-PCR and Western Blot.(2) Four different pairs of siRNA(siRNA-l,siRNA-2,siRNA-3,siRNA- 4) were designed.(3) The negative control siRNAs with fluorescence were divided into four groups according to different concentrations(25 nmol/L,50 nmol/L,75 nmol/L,100 nmol/L) and transfected into HUVEC with liposome.Fluorescent microscope was employed to determine the concentration at which siRNAs were transfected most effectively.(4) HUVEC were divided into eight groups;four groups transfected by the different siRNAs already designed,GAPDH positive control group(GAPDH-siRNA-PC),negative control group(siRNA-NC), mock control group(Mock) and blank control group (Blank).These siRNAs were transfected at the optimal concentration. After 48 hours,the one that most extremely silenced SOCS1 was selected by RT-PCR and Western Blotting. (5 )The selected siRNA as well as siRNA-NC was transfected at the optimal concentration.After 24 hours,HUVEC were divided into four groups:a.siRNA-NC;b.siRNA-NC + hypoxia/reoxygenation;c.SOCS1 iRNA;d.SOCS1 siRNA+hypoxia/ reoxygenation.Then,the expressions of Caspase-3 and Bax were detected by Western Blotting,and the apoptosis rates were assessed by flow cytometry.Results(1)It was 50 nmol/L at which siRNAs were transfected most effectively. (2) The expressions of SOCS1 in siRNA-1,siRNA-2, siRNA-3 and siRNA-4 groups declined compared to four control groups(P<0.05).SiRNA-3 had the optimal silencing efficiency in four different pairs of siRNA(P<0.05).(3) After RNAi and hypoxia/reoxygenation,the expressions of Caspase-3 and Bax in SOCS1 siRNA+hypoxia/reoxygenation group elevated compared to other three groups(P<0.05).The expressions of Caspase-3 and Bax in siRNA-NC+hypoxia/re-oxygenation group increased compared to siRNA-NC group (P<0.05).The expression of Caspase-3 in siRNA-NC+hypoxia/ reoxygenation group increased compared to SOCS1 siRNA group(P<0.05).There was no statistical significance of Caspase-3 between siRNA-NC and SOCS1 siRNA groups(P> 0.05).Bax in SOCS1 siRNA group was higher than that in siRNA-NC group(P<0.05).(4) After RNAi and hypoxia/reoxygenation, the apoptosis rate of SOCS1 siRNA+hypoxia/re-oxygenation group increased compared to other three groups (P<0.05).The apoptosis rate of siRNA-NC+hypoxia/reoxy-genation group increased compared to siRNA-NC and SOCS1 siRNA groups(P<0.05).There was no statistical significance between siRNA-NC and SOCS1 siRNA groups(P>0.05). Conclusions(1) The expression of SOCS1 in EC could be inhibited effectively by RNAi.(2)SOCS1 silencing could exacerbate EC apoptosis induced by hypoxia/reoxygenation.更多还原