HIF-1α介导大鼠血管内皮细胞ET-1及其受体表达的机制研究

Hypoxia inducible factor-1 mediates the transactivation of endothelin-1 and its receptors in rat endothelial cells

目的探讨低氧诱导因子-1(HIF-1)在大鼠血管内皮细胞中介导内皮素-1(ET-1)及其受体表达的作用及机制。方法在大鼠内皮细胞中过表达或siRNA干扰HIF-1α的表达,过表达分为空白组、GV230组、GV230/HIF-1α组,siRNA干扰分为空白组、shRNA-NC组、shRNA/HIF-1α组。应用Real-time PCR、Western blot法检测HIF-1α、ET-1、ETA、ETB的mRNA和蛋白表达水平。采用2^(-ΔΔCt)计算Real-time PCR的基因表达水平,采用GraphPad Prism 5.0软件对结果进行统计处理。采用Quantity one灰度分析软件分析Western blot的蛋白表达水平。应用单因素的方差分析比较多组之间均数的差异,各组之间的两两比较采用LSD法。结果与空白组和GV230组相比,GV230/HIF-1α组的细胞中ET-1、内皮素受体A (ETA)、内皮素受体B(ETB)mRNA和蛋白表达水平显著增高,差异有统计学意义(P<0.05);与空白组和shRNA-NC组相比,shRNA/HIF-1α组的细胞中ET-1、ETA、ETB mRNA和蛋白表达水平显著降低,差异有统计学意义(P<0.05)。空白组和GV230组/shRNA-NC组的ET-1、ETA、ETB mRNA和蛋白表达水平差异无统计学意义(P>0.05)。结论 HIF-1α介导血管内皮细胞中ET-1及其受体表达,参与低氧条件下血管通透性变化。

Objective To examine the molecular mechanism of hypoxia inducible factor-1 mediates the transactivation of endothelin-1(ET-1)and its receptors in rat endothelial cells.Methods Hypoxia-inducible factor 1 alpha(HIF-1α)was over-expressed or depleted in the rat endothelial cells.Overexpression was divided into blank group,GV230 group,GV230/HIF-1αgroup,siRNA interference was divided into blank group,shRNA-NC group,shRNA/HIF-1αgroup.Real-time PCR and Western blot were usded to detect the mRNA and protein levels of HIF-1α,ET-1,endothelin receptor A(ETA)and endothelin receptor B(ETB).The mRNA expression level was estimated by 2-ΔΔCtand analyzed by GraphPad Prism 5.0,and the protein expression level was analyzed by Quantity one.The differences among groups were analyzed by One-Way ANOVA method and LSD.Results The mRNA and protein levels of HIF-1α,ET-1,ETA and ETB were significantly higher in the GV230/HIF-1αgroup than that in the blank control group and the GV230 group.The difference was statistically significant(P<0.05).However,the mRNA and protein levels of HIF-1α,ET-1,ETA and ETB were significantly lower in the shRNA/HIF-1αgroup than that in the blank control group and the shRNA-NC group.The difference was statistically significant(P<0.05).There were no significant differences on the mRNA and protein levels of HIF-1α,ET-1,ETA and ETB between the blank control group and the GV230 group or the shRNA-NC group(P>0.05).Conclusion HIF-1αmediates the expression of ET-1 and its receptors in vascular endothelial cells and participates in the changes of vascular permeability under hypoxic conditions.