白藜芦醇延缓心肌微血管内皮细胞衰老的作用机制  

Mechanism of Resveratrol in Delaying Senescence of Cardiac Microvascular Endothelial Cells

作　　者：王强[1] 修成奎[2] 雷燕[2] 杨静[2] WANG Qiang;XIU Cheng-kui;LEI Yan;YANG Jing(Wangjing Hospital,China Academy of Chinese Medical Sciences,Beijing 100102,China;Experimental Research Center,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]中国中医科学院望京医院,北京100102 [2]中国中医科学院医学实验中心,北京100700

出　　处：《中国实验方剂学杂志》2018年第19期145-152,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81273976,81703865);中国中医科学院自主选题项目(ZZ2013002)

摘　　要：目的：探讨白藜芦醇延缓人心脏微血管内皮细胞衰老的作用,从细胞骨架角度分析其作用机制。方法：通过热休克蛋白27（HSP27）shRNA细胞系的建立,将组别为8代复制性衰老模型组（衰老组）,白藜芦醇组（10μmol·L-1）,空载质粒（Mock）组,HSP27shRNA组,HSP27shRNA＋白藜芦醇组（10μmol·L-1）。运用β-半乳糖苷酶染色检测细胞衰老,细胞计数（cell counting kit-8,CCK-8）检测细胞增殖能力,激光免疫共聚焦显微镜观察纤维状肌动蛋白（F-actin）和球型蛋白单体（G-actin）的形态学改变,蛋白免疫印迹法（Western blot）检测F-actin蛋白的表达情况。结果：与衰老组比较,白藜芦醇组β-半乳糖苷酶染色比例下降（P〈0.01）,细胞增殖能力增加（P〈0.01）,F-actin线条较为完整的分布于细胞边缘,G-actin圆润饱满的分布于细胞中央,蛋白表达减少（P〈0.01）;热休克蛋白27（heat shock protein 27,HSP27）沉默后,与Mock组比较,HSP27shRNA组及HSP27shRNA＋白藜芦醇组β-半乳糖苷酶染色比例增加（P〈0.01）,细胞增殖能力下降（P〈0.01）,F-actin外周致密带边缘变得毛糙不规整,崩解消散,细胞的形状不能清晰的呈现;G-actin表现为边缘变模糊呈絮状向外延伸,而细胞中央区域染色离散,形态不规则,F/G-actin的平均光密度值下降（P〈0.01）,蛋白表达减少（P〈0.01）。结论：白藜芦醇能够延缓内皮衰老,促进增殖,其中HSP27下调F-actin的表达可能是白藜芦醇延缓内皮衰老的重要机制所在。Objective： To investigate the effect of resveratrol in delaying senescence of human cardiac microvascular endothelial cells（ HCMEC）, and to analyze its mechanism of action from the perspective of cytoskeleton. Method： Heat shock protein 27（ HSP27） shRNA cell lines were established,and the cells were divided into 5 groups,the eighth generation replicative senescence model group（ senescence group）,resveratrol group（ 10 μmol·L-1）,mock group,HSP27 shRNA group,HSP27 shRNA ＋ resveratrol group（ 10 μmol·L-1）.Cells senescence was detected by using β-galactosidase staining; cell counting kit-8（ CCK-8） was used to detect cell proliferation; laser confocal microscopy was used to observe the morphological changes of fibrous actin（ F-actin） and globular actin（ G-actin）. Western blot was used to detect the protein expression of F-actin. Result：As compared with the senescence group,resveratrol could decrease the proportion of β-galactosidase stained（ P〈 0. 01）,promote cell proliferation（ P〈 0. 01）,maintain F-actin distribution in the cell periphery and G-actin distribution in the cell center,and reduce the protein expression（ P〈 0. 01）. After silencing of heat shock protein27（ HSP27）,as compared with mock group,HSP27 shRNA group and HSP27 shRNA ＋ resveratrol group could increase the proportion of β-galactosidase stained（ P 〈0. 01）,decrease cell proliferation（ P 〈0. 01）; F-actin around the cell periphery became irregular,sputtered and dissipated gradually,with unclear cell shape; G-actin showed blurred edges and stretched outside in cotton-like method,with staining dispersion in cell central region and irregular shape. The mean optical density value of F/G-actin was decreased（ P 〈0. 01）,and the protein expression was also decreased（ P〈 0. 01）. Conclusion： Resveratrol could delay endothelial senescence,promote cell proliferation,and the mechanism may be associated with down-regulation of the F-actin expression by HSP27.

关 键 词：白藜芦醇 内皮衰老 heat shock protein 27(HSP27)shRNA细胞素 纤维状肌动蛋白 

分 类 号：R2-0[医药卫生—中医学] R22