HIF-1α对大鼠内皮细胞通透性的影响及作用机制  

Effects and mechanism of HIF-1α on the permeability of rat endothelial cells

作　　者：蒋智永 胡子健 孟承颖 黄海良 段声梁 蒋薇 王欢[1] 余又新[1] 孙业祥[1] 方林森[1] 胡德林[1] Jiang Zhiyong;Hu Zijian;Meng Chengying(Dept of Burn, The First Affiliated Hospital of Anhui Medical University, Hefei 230022;Dept of Clinical Medicine, First Clinical Medical College, Anhui Medical University, Hefei 230022)

机构地区：[1]安徽医科大学第一附属医院烧伤科,合肥230022 [2]安徽医科大学第一临床医学院,合肥230022 [3]安徽医科大学基础医学院生物化学与分子生物学教研室,合肥230022

出　　处：《安徽医科大学学报》2019年第7期1061-1065,共5页Acta Universitatis Medicinalis Anhui

基　　金：2015年度第二批安徽省自然科学基金项目(编号:1508085SMH231);安徽省2018年度重点研究与开发计划项目(编号:1804h08020230)

摘　　要：目的探讨低氧诱导因子-1 (HIF-1α)对大鼠主动脉内皮细胞通透性的影响及作用机制。方法构建HIF-1α干扰及过表达载体,应用Lipofectamine 2000转染原代大鼠主动脉内皮细胞,浓度为200μg/ml遗传霉素(G418)筛选稳转细胞株,实时荧光定量PCR(RT-PCR)和Western blot检测HIF-1α、内皮素-1(ET-1)、内皮素受体A(ETA)、内皮素受体B(ETB)和闭锁小带(ZO-1)的表达;应用ET-1信号通路抑制剂BQ123及BQ788预处理HIF-1α过表达细胞株1 h,RT-PCR和Western blot检测HIF-1α、ET-1、ETA、ETB和ZO-1的表达,细胞膜通透性实验检测单层内皮细胞膜通透系数变化。结果与转染短发夹RNA-阴性对照组(shRNA-NC组)相比,转染shRNA-Ⅱ组HIF-1α、ET-1、ETA、ETB mRNA和蛋白表达水平均显著降低(P<0.01),ZO-1的表达则明显升高(P<0.01)。与转染GV230组比较,稳转GV230/HIF-1α组HIF-1α、ET-1、ETA、ETB mRNA和蛋白表达水平均显著升高(P<0.01),ZO-1的表达则明显降低(P<0.01);同时细胞膜通透性系数亦明显升高(P<0.01);应用BQ123和BQ788处理能够抑制HIF-1α诱导的ETA和ETB表达、增强ZO-1的表达,同时细胞膜通透性系数明显降低(P<0.01)。结论 HIF-1α可通过上调ET-1的表达进而增加大鼠主动脉内皮细胞的通透性,HIF-1α/ET-1信号轴在调控烧伤大鼠血管通透性方面具有重要作用。Objective To investigate the effects and mechanism of hypoxia-inducible factor 1α(HIF-1α) on the permeability of rat aortic endothelial cells. Methods Interference and overexpression vector of HIF-1α was transfectant into rat aortic endothelial cells by Lipofectamine 2000 respectively. Rat aortic endothelial cells were cultured at a concentration of 200 μg/ml G418. The expressions of HIF-1α, endothelin-1(ET-1),endothelin receptor A(ETA),endothelin receptor B(ETB) and zonula occludens(ZO-1) were detected by Real-time PCR and Western blot. The signal transduction inhibitor BQ123 and BQ788 was applied to discover the activity changes of HIF-1α/ET-1 sigmal. Meanwhlie, the membrane permeability coefficient of the monolayer endothelial cells was examined. Results Compared with the control group, the levels of HIF-1α and ET-1 in the scalded rats were significantly increased(P<0.01, P<0.01). Compared with the control group, the mRNA and protein expression levels of HIF-1α, ET-1, ETA and ETB were significantly decreased in the transfected HIF-1α interfered group(P<0.01), while the expression of ZO-1 was obviously increased(P<0.01). The results of transfection of the HIF-1α overexpression vector group were reversed. At the same time, the cell membrane permeability coefficient was significantly increased(P<0.01), and the cell membrane permeability coefficient was decreased after treatment with BQ123 and BQ788. It could inhibit the expression of ETA and ETB induced by HIF-1α and enhance the expression of ZO-1, and the permeability coefficient of cell membrane was significantly decreased(P<0.01).Conclusion HIF-1α can increase the permeability of rat aortic endothelial cells by up-regulating the expression of ET-1, and the HIF-1α/ET-1 signal axis plays an important role in regulating vascular permeability in burned rats.

关 键 词：大鼠 内皮细胞 血管通透性 缺氧诱导因子-1Α 

分 类 号：R332[医药卫生—人体生理学] R977.6[医药卫生—基础医学]