Hu D L, Yu Y X, Liang R, Zhou S Y, Duan S L, Jiang Z Y, Meng C Y, Jiang W, Wang H, Sun Y X, Fang L S
Department of Burns, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China.
Department of Burns, Health-center of Shangpai Town, Feixi County, Anhui Province, Feixi 231200, China.
Zhonghua Shao Shang Za Zhi. 2019 Mar 20;35(3):209-217. doi: 10.3760/cma.j.issn.1009-2587.2019.03.009.
To investigate the regulation of hypoxia-inducible factor-1α (HIF-1α) on permeability of rat vascular endothelial cells and the mechanism. Twelve male Sprague-Dawley rats aged 35 to 38 days were collected and vascular endothelial cells were separated and cultured. The morphology of cells was observed after 4 days of culture, and the following experiments were performed on the 2nd or 3rd passage of cells. (1) Rat vascular endothelial cells were collected and divided into blank control group, negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group according to the random number table (the same grouping method below), with 3 wells in each group. Cells in negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group were transfected with GV248 empty plasmid, recombinant plasmid respectively containing HIF-1α interference sequence 1, interference sequence 2, and interference sequence 3 with liposome 2000. Cells in blank control group were only transfected with liposome 2000. After transfection of 24 h, expression levels of HIF-1α mRNA and protein of cells in each group were respectively detected by reverse transcription real-time fluorescent quantitative polymerase chain reaction and Western blotting (the same detecting methods below) . The sequence with the highest interference efficiency was selected. (2) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α low expression group, with 3 wells in each group. Cells in blank control group were only transfected with liposome 2000, and cells in negative control group and HIF-1α low expression group were respectively transfected with GV248 empty plasmid and low expression HIF-1α recombinant plasmid selected in experiment (1) with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α in each group were detected. (3) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α high expression group, with 3 wells in each group. Cells in blank control group were transfected with liposome 2000, and cells in negative control group and HIF-1α high expression group were respectively transfected with GV230 empty plasmid and HIF-1α high expression recombinant plasmid with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α of cells in each group were detected. (4) After transfection of 24 h, cells of three groups in experiment (1) and three groups in experiment (2) were collected, and mRNA and protein expressions of myosin light chain kinase (MLCK), phosphorylated myosin light chain (p-MLC), and zonula occludens 1 (ZO-1) of cells were detected. Data were processed with one-way analysis of variance and test. After 4 days of culture, the cells were spindle-shaped, and rat vascular endothelial cells were successfully cultured. (1) The interference efficiencies of HIF-1α of cells in HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group were 47.66%, 45.79%, and 62.62%, respectively, and the interference sequence 3 group had the highest interference efficiency. After transfection of 24 h, the mRNA and protein expression levels of HIF-1α of cells in interference sequence 3 group were significantly lower than those in blank control group (=18.404, 9.140, <0.01) and negative control group (=15.099, 7.096, <0.01). (2) After cultured for 14 days, the mRNA and protein expression levels of HIF-1α of cells in HIF-1α low expression group were significantly lower than those in blank control group (=21.140, 5.440, <0.01) and negative control group (= 14.310, 5.210, <0.01). (3) After cultured for 14 days, the mRNA and protein expression levels of HIF-1α of cells in HIF-1α high expression group were significantly higher than those in blank control group (=19.160, 7.710, <0.01) and negative control group (= 19.890, 7.500, <0.01). (4) After transfection of 24 h, the mRNA expression levels of MLCK and p-MLC of cells in HIF-1α low expression group were significantly lower than those in blank control group (=2.709, 4.011, <0.05 or <0.01) and negative control group (=2.373, 3.744, <0.05 or <0.01). The mRNA expression level of ZO-1 of cells in HIF-1α low expression group was significantly higher than that in blank control group and negative control group (=4.285, 5.050, <0.01). The mRNA expression levels of MLCK and p-MLC of cells in HIF-1α high expression group were significantly higher than those in blank control group (=9.118, 11.313, <0.01) and negative control group (=9.073, 11.280, <0.01). The mRNA expression level of ZO-1 of cells in HIF-1α high expression group was significantly lower than that in blank control group and negative control group (=2.889, 2.640, <0.05). (5) After transfection of 24 h, the protein expression levels of MLCK and p-MLC of cells in HIF-1α low expression group were significantly lower than those in blank control group (=2.652, 3.983, <0.05 or <0.01) and negative control group (=2.792, 4.065, <0.05 or <0.01). The protein expression of ZO-1 of cells in HIF-1α low expression group was significantly higher than that in blank control group and negative control group (=3.881, 3.570, <0.01). The protein expression levels of MLCK and p-MLC of cells in HIF-1α high expression group were 1.18±0.24 and 0.68±0.22, which were significantly higher than 0.41±0.21 and 0.35±0.14 in blank control group (=5.011, 3.982, <0.05 or <0.01) and 0.43±0.20 and 0.36±0.12 in negative control group (= 4.880, 3.862, <0.05 or <0.01). The protein expression level of ZO-1 of cells in HIF-1α high expression group was 0.08±0.06, which was significantly lower than 0.20±0.09 in blank control group and 0.19±0.09 in negative control group (=4.178, 3.830, <0.05 or <0.01). HIF-1α up-regulates expressions of MLCK and p-MLC and down-regulates expression of ZO-1, thereby increasing the permeability of rat vascular endothelial cells.
探讨缺氧诱导因子-1α（HIF-1α）对大鼠血管内皮细胞通透性的调控及其机制。选取12只35至38日龄的雄性Sprague-Dawley大鼠，分离并培养血管内皮细胞。培养4天后观察细胞形态，并在细胞传代至第2代或第3代时进行以下实验。（1）收集大鼠血管内皮细胞，按随机数字表法分为空白对照组、阴性对照组、HIF-1α干扰序列1组、HIF-1α干扰序列2组和HIF-1α干扰序列3组（以下分组方法相同），每组3孔。阴性对照组、HIF-1α干扰序列1组、HIF-1α干扰序列2组和HIF-1α干扰序列3组细胞分别用脂质体2000转染GV248空质粒、分别含HIF-1α干扰序列1、干扰序列2和干扰序列3的重组质粒。空白对照组细胞仅用脂质体2000转染。转染24小时后，分别采用逆转录实时荧光定量聚合酶链反应和蛋白质印迹法（以下检测方法相同）检测各组细胞中HIF-1α mRNA和蛋白的表达水平。选取干扰效率最高的序列。（2）另取一批大鼠血管内皮细胞，分为空白对照组、阴性对照组和HIF-1α低表达组，每组3孔。空白对照组细胞仅用脂质体2000转染，阴性对照组和HIF-1α低表达组细胞分别用脂质体2000转染实验（1）中筛选出的GV248空质粒和HIF-1α低表达重组质粒。培养14天后，检测各组细胞中HIF-1α的mRNA和蛋白表达。（3）另取一批大鼠血管内皮细胞，分为空白对照组、阴性对照组和HIF-1α高表达组，每组3孔。空白对照组细胞用脂质体2000转染，阴性对照组和HIF-1α高表达组细胞分别用脂质体2000转染GV230空质粒和HIF-1α高表达重组质粒。培养14天后，检测各组细胞中HIF-1α的mRNA和蛋白表达。（4）转染24小时后，收集实验（1）中的3组细胞和实验（2）中的3组细胞，检测细胞中肌球蛋白轻链激酶（MLCK）、磷酸化肌球蛋白轻链（p-MLC）和紧密连接蛋白1（ZO-1）的mRNA和蛋白表达。数据采用单因素方差分析和检验进行处理。培养4天后，细胞呈纺锤形，成功培养出大鼠血管内皮细胞。（1）HIF-1α干扰序列1组、HIF-1α干扰序列2组和HIF-1α干扰序列3组细胞中HIF-1α的干扰效率分别为47.66%、45.79%和62.62%，其中干扰序列3组干扰效率最高。转染24小时后，干扰序列3组细胞中HIF-1α的mRNA和蛋白表达水平显著低于空白对照组（=18.404，9.140，<0.01）和阴性对照组（=15.099，7.096，<0.01）。（2）培养14天后，HIF-1α低表达组细胞中HIF-1α的mRNA和蛋白表达水平显著低于空白对照组（=21.140，5.440，<0.01）和阴性对照组（=14.310，5.210，<0.01）。（3）培养14天后，HIF-1α高表达组细胞中HIF-1α的mRNA和蛋白表达水平显著高于空白对照组（=19.160，7.710，<0.01）和阴性对照组（=19.890，7.5用0，<0.01）。（4）转染24小时后，HIF-1α低表达组细胞中MLCK和p-MLC的mRNA表达水平显著低于空白对照组（=2.709，4.011，<0.05或<0.01）和阴性对照组（=2.373，3.744，<0.05或<0.01）。HIF-1α低表达组细胞中ZO-1的mRNA表达水平显著高于空白对照组和阴性对照组（=4.285用，5.050，<0.01）。HIF-1α高表达组细胞中MLCK和p-MLC的mRNA表达水平显著高于空白对照组（=9.118，11.313，<0.01）和阴性对照组（=9.073，11.280，<0.01）。HIF-1α高表达组细胞中ZO-1的mRNA表达水平显著低于空白对照组和阴性对照组（=2.889，2.640，<0.05）。（5）转染24小时后，HIF-1α低表达组细胞中MLCK和p-MLC的蛋白表达水平显著低于空白对照组（=2.652，3.983，<0.05或<0.01）和阴性对照组（=2.792，4.065，<0.05或<0.01）。HIF-1α低表达组细胞中ZO-1的蛋白表达显著高于空白对照组和阴性对照组（=3.881，3.570，<0.01）。HIF-1α高表达组细胞中MLCK和p-MLC蛋白水平分别为1.18±0.24用和0.68±0.22，显著高于空白对照组的0.41±0.21用和0.35±0.14（=5.011，3.982，<0.05或<0.01）和阴性对照组的0.43±0.20用和0.36±0.12（=4.880，3.862，<0.05或<0.01）。HIF-1α高表达组细胞中ZO-1蛋白水平为0.08±0.06，显著低于空白对照组的0.20±0.09用和阴性对照组的0.19±0.09（=4.178，3.830，<0.05或<0.01）。HIF-1α上调MLCK和p-MLC的表达，下调ZO-1的表达，从而增加大鼠血管内皮细胞的通透性。
