摘要
目的探讨微小RNA(miRNA)-335对缺氧复氧(H/R)诱导下大鼠心肌微血管内皮细胞(CMEC)凋亡的作用及其机制。方法建立大鼠CMEC H/R模型。采用体外合成的大鼠miR-335的模拟物和抑制剂,用脂质体转染培养的大鼠CMEC 48h后,H/R处理细胞。实验分为4组:对照组(CTL组)、H/R组、H/R加miR-335模拟物组(mimic组)、H/R加miR-335抑制剂组(inhibitor组)。采用超氧化物阴离子荧光探针法观察细胞内超氧阴离子(O2·-)的变化水平、JC-1试剂盒检测细胞线粒体膜电位的变化,流式细胞仪检测各组细胞凋亡。结果在H/R处理后,H/R组超氧化物歧化酶(SOD2)蛋白表达明显下调,而miR-335的表达明显上调。与CTL组比较,H/R组、mimic组、inhibitor组O2·-水平[(35.23±3.21)%、(46.71±2.58)%、(15.9±2.13)%vs(4.46±0.98)%,P<0.01]、细胞凋亡率[(30.46±1.63)%、(40.21±1.73)%、(17.83±1.45)%vs(2.14±0.33)%,P<0.01]明显上升,橙光与绿光比值[(0.47±0.03)、(0.28±0.05)、(0.66±0.03)vs(1.0±0.07),P<0.01]显著下降。结论miR-335能够增加氧化应激条件下心肌微血管内皮细胞的凋亡,miR-335通过调控SOD2蛋白的表达,在缺氧复氧介导的线粒体凋亡途径中扮演了关键性的作用。
Objective To study the role of miR-335 in regulating hypoxia/reoxygenation(H/R)-induced apoptosis of cardiac microvascular endothelial cells(CMEC)and its mechanism.MethodsSuperoxide dismutase 2(SOD2)and miR-335 expression levels were detected.After a rat H/R model was established.Rat CMEC were divided into control group,H/R group,H/R+ mimic group,and H/R+inhibitor group after they were transfected with miR-335 mimics or inhibitors and cultured for 48 h.Intracellular superoxide anion was detected with a superoxide anion fluorescence probe,mitochondrial membrane potential was measured with a JC-1kit,and apoptosis of CMEC was assayed by flow cytometry.Resluts The SOD2 expression level was significantly lower whereas the miR-335 expression level was significantly higher in H/R group after H/R treatment than before H/R treatment(P〈0.05).The production of superoxide anion and the apoptosis rate of CMEC were significantly higher whereas the ratio of orange/green fluorescence was lower in H/R group,H/R+mimic group,H/R+inhibitor group than in control group(35.23%±3.21%,46.71%±2.58%,15.9% ±2.13% vs4.46% ±0.98%,P〈0.01;30.46% ±1.63%,40.21%±1.73%,17.83%±1.45%vs 2.14%±0.33%,P〈0.01;0.47±0.03,0.28±0.05,0.66±0.03 vs 1.0±0.07,P〈0.01).Conclusion MiR-335 playing a key role in H/R-induced mitochondrial apoptosis under oxidative stress condition.
出处
《中华老年心脑血管病杂志》
CAS
2016年第7期739-743,共5页
Chinese Journal of Geriatric Heart,Brain and Vessel Diseases
基金
湖北省教育厅科学研究项目(Q20152104)