上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

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

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   

  1. 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
  • 出版日期:2015-05-28 发布日期:2015-06-04
  • 通讯作者: 李颖川, 电子信箱: yingchuan.li@sjtu.edu.cn
  • 作者简介:姜桢（1944—）， 女， 主任医师; 电子信箱: jiangzhen66@yahoo.com.cn。
  • 基金资助:

    浦东新区科技发展基金（PKj2011-y36）

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

JIANG Zhen1, CAO Yong-mei2, ZENG Zhen2, GU Yu-chun1, JI Ying-ying1, ZHAO Yu-peng1, LI Ying-chuan2   

  1. 1.Department of Anesthesiology, Dongfang Hospital, Tongji University, Shanghai 200120, China; 2.Department of Anesthesiology, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai 200233, China
  • Online:2015-05-28 Published:2015-06-04
  • Supported by:

    Pudong New Area Science and Technology Development Fund, PKj2011-y36

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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。

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

Abstract:

Objective To investigate the effects of hypoxia response element (HRE)-mediated Bcl-2 gene silencing on hypoxia-induced anti-apoptotic pulmonary vascular endothelial cells. Methods The siRNA sequence for Bcl-2 was designed. Bcl-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 significantly 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 Bcl-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.

Key words: Bcl-2, hypoxia response element, anti-apoptotic pulmonary vascular endothelial cells, gene regulation