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
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摘要
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-1α group,siRNA interference was divided into blank group,shRNA-NC group,shRNA/HIF-1α group. 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-1α group 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-1α group 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-1α mediates the expression of ET-1 and its receptors in vascular endothelial cells and participates in the changes of vascular permeability under hypoxic conditions.
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-1α group,siRNA interference was divided into blank group,shRNA-NC group,shRNA/HIF-1α group. 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-1α group 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-1α group 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-1α mediates the expression of ET-1 and its receptors in vascular endothelial cells and participates in the changes of vascular permeability under hypoxic conditions.
引文
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[14] 杨晓菊,孙玲玲,张萌,等.糖尿病患者妊娠后胎盘组织缺氧诱导因子-1α及内皮素-1的表达及与妊娠结局关系[J].中华实用诊断与治疗杂志,2014, 28(1):15-7.
[15] 王三应,王国付,毛根祥,等.内皮素-1与心脑血管疾病研究进展[J].心脑血管病防治,2016,16(4):291-3.
[2] Yegutkin G G, Helenius M, Kaczmarek E, et al. Chronic hypoxia impairs extracellular nucleotide metabolism and barrier function in pulmonary artery vasa vasorum endothelial cells[J]. Angiogenesis, 2011, 14(4): 503-13.
[3] 赵荣瑞.缺氧诱导因子-1(HIF-1)的基础研究与临床意义[J].山西医科大学学报,2006,37(3):225-30.
[4] Andrikopoulos P, Kieswich J, Harwood S M, et al. Endothelial angiogenesis and barrier function in response to thrombin require Ca2+ influx through the Na+/Ca2+ exchanger[J]. J Biol Chem, 2015, 290(30): 18412-28.
[5] 张晓军,刘健,万磊,等. 佐剂关节炎大鼠滑膜组织HIF-1α和VEGF及CD34表达上调促进血管新生[J]. 细胞与分子免疫学杂志,2015, 31(8): 1053-6.
[6] Cox R A, Enkhabaatar P, Burke A S, et al. Effects of a dual endothelin-1 receptor antagonist on airway obstruction and acute lung injury in sheep following smoke inhalation and burn injury[J]. Clin Sci(Lond), 2005, 108(3): 265-72.
[7] Suzuki N, Gradin K, Poellinger L, et al. Regulation of hypoxia-inducible gene expression after HIF activation[J]. Exp Cell Res, 2017, 356(2):182-6.
[8] 台运成. HIF-1/ET-1途径对大鼠血管内皮细胞通透性的调控作用及机制[D].合肥:安徽医科大学,2016.
[9] 孙改霞,郭亚雄,杜会博,等. 失血性休克后的肠淋巴液提高血管通透性的作用[J]. 中国病理生理杂志,2014,30(8):1506-12, 1536.
[10] Spinella F, Rosanò L, Del Duca M, et al.Endothelin-1 inhibits prolyl hydroxylase domain 2 to activate hypoxia-inducible factor-1alpha in melanoma cells[J]. PLoS One, 2010, 5(6): e11241.
[11] Caprara V,Scappa S,Garrafa E,et al.Endothelin-1 regulates hypoxia-inducible factor-1alpha and -2alpha stability through prolyl hydroxylase domain 2 inhibition in human lymphatic endothelial cells[J].Life Sci,2014,118(2): 185-90.
[12] 戴跃龙,胡森,窦永起.烧伤早期血管内皮屏障损伤机制与保护药物的研究进展[J].感染、炎症、修复,2016,17(4):248-50.
[13] 屈莉红,荆鲁宁.胎盘组织缺氧诱导因子-1α和内皮素-1表达对糖尿病妊娠的影响[J].中国现代医学杂志,2016,26(20):44-7.
[14] 杨晓菊,孙玲玲,张萌,等.糖尿病患者妊娠后胎盘组织缺氧诱导因子-1α及内皮素-1的表达及与妊娠结局关系[J].中华实用诊断与治疗杂志,2014, 28(1):15-7.
[15] 王三应,王国付,毛根祥,等.内皮素-1与心脑血管疾病研究进展[J].心脑血管病防治,2016,16(4):291-3.