小干扰RNA下调cyclophilin-D蛋白表达对内皮细胞抗氧化损伤的作用☆

背景：线粒体基质中的Cyclophilin-D蛋白(CypD)在线粒体损伤过程中起着特殊作用。目前对于CypD蛋白与血管内皮细胞功能的关系，以及CypD蛋白在动脉粥样硬化和血管狭窄发生、发展中的作用仍未明确。 目的：观察小干扰RNA基因沉默对内皮细胞CyPD蛋白表达及氧化应激损伤、凋亡的影响。 方法：采用含500 μmol/L H2O2的PBS液处理ECV304细胞，小干扰RNA沉默ppif基因以建立CyPD蛋白缺陷型细胞凋亡模型，空白对照组以单纯PBS液处理。流式细胞仪检测ECV304细胞凋亡率，电镜观察细胞超微结构。 结果与结论：CyPD缺陷模型组细胞凋亡率为(32.51±6.6)%，明显低于500 μmol/L H2O2处理组(52.57±5.84)%，P=0.001。电镜观察结果显示，CyPD缺陷型细胞模型组细胞凋亡数量明显较500 μmol/L H2O2处理组减少，形态改变也较轻，多为早期凋亡特征。提示下调CyPD蛋白水平可明显减轻血管内皮细胞氧化应激损伤和凋亡。关键词：内皮细胞；氧化应激；RNA干扰；cyclophilin-D蛋白；线粒体缩 缩略语注释：siRNA: small interfering RNA，小干扰RNA doi:10.3969/j.issn.1673-8225.2012.15.032

Effect of small interfering RNA down-regulated Cyclophilin-D protein expression against oxidative injury to endothelial cells

BACKGROUND: Cyclophilin-D protein in the mitochondrial matrix plays a special role in the process of mitochondrial damage. Correlation between Cyclophilin-D protein and vascular endothelial cell function, as well as Cyclophilin-D protein effects on atherosclerosis and vascular stenosis are still unclear. OBJECTIVE: To investigate the relationship between Cyclophilin-D expression and oxidative stress damage to endothelial cells by small interfering RNA (siRNA)-mediated gene silencing. METHODS: ECV304 cells were cultured in phosphate buffered saline containing 500 μmol/L H2O2. siRNA silenced ppif gene was used to establish Cyclophilin-D deficiency models. The blank group was treated with phosphate buffered saline. Apoptotic rate of ECV304 cells was detected by flow cytometry; meanwhile, morphological changes were observed by electron microscope. RESULTS AND CONCLUSION: The apoptotic rate of ECV304 cells was (32.51±6.6)% in the Cyclophilin-D deficiency group, significantly lower than (52.57±5.84)% in the H2O2 group (P=0.001). The electron microscope results showed that the number of apoptotic cells in the Cyclophilin-D deficiency group was decreased significantly as compared with the H2O2 group, and the morphological changes in the former group were slighter that were characterized as early apoptosis. Cyclophilin-D is a key protein to vascular endothelial cells against oxidative stress injury.