RNA干扰抑制缺氧诱导因子1α表达对缺氧内皮细胞通透性的影响

Effect of hypoxia-inducible factor-1α inhibition caused by RNA interference on permeability of hypoxic endothelial cells

目的 了解RNA干扰技术特异性抑制缺氧诱导因子1α（HIF-1α）表达对缺氧血管内皮细胞通透性的影响.方法 利用质粒pcDNA6.2-GW/EmGFP-miR构建针对人HIF-1α基因的RNA干扰表达载体.将VE细胞分为正常对照组、缺氧组（置于含体积分数1%O_2的混合气体环境中缺氧处理6 h）、转染组、转染缺氧组（转染载体后再进行缺氧处理）.采用RT-PCR法检测正常对照组、转染组HIF-1αmRNA表达,采用蛋白质印迹法检测4组细胞HIF-1α蛋白表达.荧光分光光度计检测单层VE细胞的通透性.将上述缺氧处理替换为HIF-1α特异性诱导剂1 mmol/L二甲氧乙二酰甘氨酸（DMOG）,分为DMOG组、转染DMOG组、正常对照组、转染组,采用蛋白质印迹法观察各组HIF-1α蛋白表达.除通透性检测数据用荧光强度值表示外,其他数据用密度比值表示.实验结果进行组间两两t检验.结果 正常对照组细胞HIF-1α mRNA相对含量为0.765±0.069,转染组细胞HIF-1α mRNA相对含量为0.093±0.007,组间比较,差异有统计学意义（t=16.696,P〈0.05）.转染缺氧组细胞HIF-1α蛋白含量为0.591±0.029,显著低于缺氧组（2.612±0.259,t=13.415,P〈0.05）;转染DMOG组HIF-1α蛋白含量为0.566±0.008,显著低于DMOG组（3.243±0.551,t=6.975,P〈0.05）.缺氧组单层血管内皮细胞通透性（41.6±11.1）较正常对照组（9.4±1.5）显著升高（t=6.238,P〈0.05）,转染缺氧组单层血管内皮细胞通透性（13.3±4.5）显著低于缺氧组（t=5.430,P〈0.05）.结论 采用特异性针对HIF-1α基因的RNA干扰技术,能有效抑制内皮细胞HIF-1α表达,并明显抑制缺氧引起的血管内皮细胞通透性增强.

Objective To study the effect of hypoxia-inducible factor-1α （HIF-1α） inhibition caused by RNA interference on permeability of hypoxic vascular endothelial （ VE） cells. Methods Plas-mid pcDNA6. 2-GW/EmGKP-miR was applied to construct the RNA interference expression vector targeted to human HIF-1α gene. VE cells were divided into normal control group （ NC） , hypoxia group （H, cells were treated for hypoxia in mixed gas with 1% O_2 for 6 hours） , transfection group （T） , and transfection hypoxia group （TH, transfected with vector and treated with hypoxia）. Expression of HIF-1α mRNA in NC and T groups were determined with RT-PCR. Expression of HIF-1α protein in each group was determined with Western blot. The permeability of VE cell monolayer was detected by fluorospectrophotometer. Another sample of VE cells were divided into dimethyloxallyl glycine （DMOG） group, tranfected with DMOG group （TD） , normal control group （NC） , and transfection group （T） , with 1 mmol/L DMOG （ HIF-1α specific derivant） replacing hypoxia treatment. The expression of HIF-1α protein in each group was determined with Western blot. All data were recorded as density value ratio except for permeability data, which was recorded as fluorescence intensity value. Data were processed with t test （ pairwise comparison among groups）. Results The relative content of HIF-1α mRNA of cells in NC group （0.765 ±0.069） was significantly higher than that of cells in T group （0.093 ±0. 007, t = 16.696, P 〈0.05）. Content of HIF-1α protein of cells in TH group （0.591 ±0. 029） was significantly lower than that of cells in H group （2. 612 ±0. 259, t -13.415, P 〈0.05）. Content of HIF-la protein of cells in TD group （0. 566 ±0.008） was significantly lower than that of cells in DMOG group （3. 243 ±0. 551, t =6.975, P 〈0.05）. The permeability of cell monolayer in H group （41.6 ±11.1） was significantly higher than that of cell monolayer in NC group （9.4 ± 1.5, t =6.238, P 〈0.05）. The permeability of cell monolayer in TH group （13.3 ±4.5） was markedly lower than that of cell monolayer in H group （ t =5.430, P 〈0.05）. Conclusions The expression of HIF-1α gene in vascular endothelial cells is effectively inhibited by specific RNA interference, which significantly prevents the hypoxia-induced increase in vascular endothelial cell permeability.