低氧反应元件介导的Bcl-2-shRNA对缺氧诱导抗凋亡肺血管内皮细胞的调控作用  

Regulation effect of hypoxia response element-mediated Bcl-2-shRNA on hypoxia-induced anti-apoptotic pulmonary vascular endothelial cells

作　　者：姜桢[1] 曹永梅[2] 曾真[2] 顾玉春[1] 季莹莹[1] 赵宇鹏[1] 李颖川[2] 

机构地区：[1]同济大学附属东方医院麻醉科,上海200120 [2]上海交通大学附属第六人民医院麻醉科,上海200233

出　　处：《上海交通大学学报（医学版）》2015年第5期674-681,共8页Journal of Shanghai Jiao tong University:Medical Science

基　　金：浦东新区科技发展基金(PKj2011-y36)~~

摘　　要：目的探讨低氧反应元件(HRE)介导的Bcl-2基因沉默对缺氧诱导抗凋亡肺微血管内皮细胞的影响。方法设计针对Bcl-2的siRNA序列,构建含Bcl-2-shRNA和HRE的表达载体并进行慢病毒包装,重组病毒感染肺微血管内皮细胞,分别在常氧(21%)或低氧(5%)条件下培养;96 h后通过荧光标记物确认感染效率,Western blotting检测转染后细胞内Bcl-2蛋白表达的变化,流式细胞术检测细胞凋亡率。结果 Bcl-2基因沉默明显下调细胞内Bcl-2 mRNA和蛋白的表达;HRE启动子仅在低氧环境中活性显著增强;重组慢病毒感染肺微血管内皮细胞后96 h时的转染效率约为90%,细胞内Bcl-2蛋白表达下调,细胞凋亡增加;在低氧环境中,受HRE的调控,Lv-HRE-Bcl-2-shRNA组细胞凋亡率显著增高。结论低氧条件下,HRE作为氧敏感调控开关,可进一步增强Bcl-2-shRNA的作用,诱导肺血管内皮细胞凋亡,这将为肺动脉高压的血管重塑提供潜在的治疗靶标。Objective To investigate the effects of hypoxia response element （HRE）-mediated Bcl-2 gene silencing on hypoxia-induced and-apoptotic pulmonary vascular endothelial cells. Methods The siRNA sequence for Bd-2 was designed. Bd-2-shRNA expression vectors carrying HRE were constructed and packaged by lentivirus. Pulmonary vascular endothelial cells infected with recombinant lentivirus were cultured under the normoxic condition （21% O2） and hypoxic condition （5% O2）, respectively. The infection efficiency was observed by the fluorescent marker after 96 h. The expression of Bcl-2 protein was detected by the Western blotting and the cell apoptosis was detected by the flow cytometry. Results The silencing of Bcl-2 gene significantly down-regulated the mRNA and protein expressions of Bcl-2. The activity of HRE promoter enhanced only in hypoxic environment. The transfection efficiency was about 90% after pulmonary microvascular endothelial cells were infected with recombinant lentivirus for 96 h and the protein level of Bcl-2 decreased and cell apoptosis increased. The apoptosis rat of Lv-HRE-Bcl-2-shRNA group significandy increased because of the regulation of HRE in hypoxic environment. Conclusion HRE acts as an oxygen-sensitive regulatory switch under the hypoxic condition, which can further enhance the effects of Bd-2-shRNA gene and induce the apoptosis of pulmonary vascular endothelial cells, so as to provide a potential therapeutic target for vascular remodeling of pulmonary arterial hypertension.

关 键 词：BCL-2 低氧反应元件 抗凋亡肺血管内皮细胞 基因调控 

分 类 号：R544.1[医药卫生—心血管疾病]