目的 探讨缺氧诱导因子-2α(HIF-2α)调控缺氧环境下内皮细胞血管形成的机制.方法 采用实验研究方法.(1)细胞分组:取对数生长期人类脐静脉内皮细胞(HUVECs):细胞不做任何处理设为空白对照组,细胞转染对照shRNA设为空载体组,细胞转染HIF-2α shRNA设为HIF-2α沉默组,细胞转染HIF-2α shRNA后加入rh-Ang-2设为HIF-2α沉默联合rh-Ang-2组.(2) Western blot检测:①缺氧环境下培养0、2、4、8、12、16、20 h的HUVECs中血管生成素-2(Ang-2)和HIF-2α的蛋白表达量.②检测空白对照组、空载体组和HIF-2α沉默组HUVECs中Ang-2和HIF-2α的蛋白表达量.(3)ELISA检测:检测空白对照组、空载体组、HIF-2α沉默组HUVECs上清液中Ang-2水平.(4)小管形成实验检测:空白对照组、空载体组、HIF-2α沉默组和HIF-2α沉默联合rh-Ang-2组HUVECs中内皮细胞小管数目.(5)Transwell法检测细胞迁移:①空白对照组、空载体组、HIF-2α沉默组和HIF-2α沉默联合rh-Ang-2组HUVECs迁移细胞数目.②空白对照组、空载体组、HIF-2α沉默组和HIF-2α沉默联合rh-Ang-2组HUVECs上清液干预肝癌细胞SMMC-7721后,肝癌细胞SMMC-7721迁移数目.正态分布的计量资料以-x±s表示,重复测量数据采用重复测量的方差分析,多组间比较采用单因素方差分析并采用Dunnett's test进行组间两两比较.结果 (1) Western blot检测相关蛋白表达量:①HUVECs缺氧培养0、2、4、8、12、16、20 h细胞中Ang-2的表达量分别为0.110±0.011、0.120±0.020、0.210±0.070、0.410±0.100、0.520±0.090、0.790±0.130、1.010±0.220;HIF-2α的表达量分别为0.180±0.090、0.410 ±0.070、0.470±0.110、0.470±0.070、0.580 ±0.120、0.690 ±0.140、0.920±0.130,经缺氧处理后两种蛋白表达量均有增高,且随缺氧时间延长,表达量呈升高趋势,差异有统计学意义(F =403.550,3 265.587,P＜0.05).②空白对照组、空载体组和HIF-2α沉默组HUVECs中Ang-2的蛋白表达量分别为1.030 ±0.180、1.070 ±0.120、0.210±0.070,HIF-2α分别为0.940±0.110、0.930 ±0.190、0.170±0.021,3组上述指标比较,差异均有统计学意义(F=290.242,26.688,P＜0.05).(2)ELISA检测结果显示:空白对照组、空载体组和HIF-2α沉默组HUVECs中Ang-2表达量为(433.2±9.7)ng/L、(438.3±2.6)ng,/L、(114.6 ±4.2)ng/L,3组比较,差异有统计学意义(F=2 642.180,P ＜0.05).(3)小管形成实验结果显示:空白对照组、空载体组、HIF-2α沉默组和HIF-2α沉默联合rh-Ang-2组HUVECs中内皮细胞小管数目分别为(48.3±2.5)个、(47.4±3.1)个、(19.7±1.5)个、(38.3±2.1)个,4组比较,差异有统计学意义(F=148.196,P＜0.05).(4) Transwell法检测细胞迁移结果:①空白对照组、空载体组、HIF-2α沉默组和HIF-2α沉默联合rh-Ang-2组HUVECs中,迁移细胞数目分别为(140.3±3.5)个、(142.7±2.1)个、(42.7±3.1)个、(78.1±4.2)个,4组比较,差异有统计学意义(F=212.205,P＜0.05).②Transwell法检测4种不同上清液干预的SMMC-7721细胞迁移结果:空白对照组、空载体组、HIF-2α沉默组和HIF-2α沉默联合rh-Ang-2组上清液分别处理SMMC-7721细胞后,其细胞迁移数目分别为:(106.7±5.5)个、(102.7±6.6)个、(63.0±3.3)个、(96.7±2.1)个,4种处理方式比较,差异有统计学意义(F =55.122,P＜0.05).结论 HIF-2α可通过Ang-2调控缺氧下内皮细胞的血管形成,并通过内皮细胞分泌的Ang-2促进肝癌细胞SMMC-7721迁移.%Objective To investigate the mechanisms of hypoxia inducible factor-2 alpha (HIF-2a) regulating human umbilical vein endothelial cells (HUVECs) under hypoxic conditions.Methods The experimental study was adopted.(1) HUVECs in logarithmic growth phase were taken:HUVECs without any disposals as control group,HUVECs with shRNA transfection control as shRNA control group,HUVECs with HIF-2α shRNA transfection as HIF-2α shRNA group and HUVECs with HIF-2α shRNA transfection then added rhAng-2 as HIF-2α ± rh-Ang-2 group.(2) Western blot testing:the expressions of Ang-2 and HIF-2α proteins in HUVECs were cultured under hypoxia conditions at 0,2,4,8,12,16,20 hours,and the levels of which were detected in the control group,shRNA control group and HIF-2α shRNA group.(3) Enzyme-linked immunosorbent assay(ELISA):the level of Ang-2 protein in supernatant of HUVECs was detected in the control group,shRNA control group and HIF-2α shRNA group.(4)The amounts of endothelial cell tubes in HUVECs among the 4 groups were detected by tube formation experimental testing.(5) Transwell method was performed to detect the amounts of cells migration in HUVECs and hepatoma cells SMMC-7721 migration intervened by supernatant of HUVECs among the 4 groups.Measurement data with normal distribution were presented as x ± s,repeated measurement data were analyzed by the repeated measures ANOVA,comparison among groups and pairwise comparison were conducted respectively by the one-way ANOVA and Dunnett's test.Results (1) Western blot test:the expression levels of Ang-2 and HIF-2α proteins in HUVECs under hypoxia conditions at 0,2,4,8,12,16,20 hours were 0.110 ±0.011,0.120 ±0.020,0.210 ±0.070,0.410 ±0.100,0.520 ± 0.090,0.790±0.130 1.010 ±0.220 and 0.180 ±0.090,0.410 ±0.070,0.470 ±0.110,0.470 ±0.070,0.580 ± 0.120,0.690 ± 0.140,0.920 ± 0.130,respectively,and which were increased after culturing under hypoxia conditions and had an ascending tendency as the hypoxia time extended,with statistically significant differences (F =403.550,3 265.587,P ＜ 0.05).The expression levels of Ang-2 and HIF-2α proteins in the control group,shRNA control group and HIF-2α shRNA group were 1.030 ±0.180,1.070 ±0.120,0.210 ± 0.070,and 0.940 ± 0.110,0.930 ± 0.190,0.170 ± 0.021,respectively,showing statistically significant differences (F =290.242,26.688,P ＜ 0.05).(2) The results of ELISA:the expression levels of Ang-2 in the control group,shRNA control group and HIF-2α shRNA group were (433.2 ±9.7)ng/L,(438.3 ± 2.6)ng/L,(114.6 ± 4.2) ng/L,with a statistically significant difference (F =2 642.180,P ＜ 0.05).(3) The results of tube formation experiments:the number of endothelial cell tubes in the control group,shRNA control group,HIF-2α shRNA group and HIF-2α ± rh-Ang-2 group were 48.3 ± 2.5,47.4 ± 3.1,19.7 ± 1.5 and 38.3 ± 2.1,respectively,with a statistically significant difference (F =148.196,P ＜ 0.05).(4) The results of Transwell method:① the number of HUVECs migration in the control group,shRNA control group,HIF-2α shRNA group and HIF-2α + rh-Ang-2 group were 140.3-± 3.5,142.7 ± 2.1,42.7 ± 3.1 and 78.1 ± 4.2,respectively,showing a statistically significant differences (F =212.205,P ＜ 0.05).②The results of Transwell method:the number of SMMC-7721 cells migration after intervening using four different supernatant in the control group,shRNA control group,HIF-2α shRNA group and HIF-2α ± rh-Ang-2 group were 106.7 ± 5.5,102.7 ± 6.6,63.0 ± 3.3 and 96.7 ± 2.1,respectively,showing a statistically significant difference (F =55.122,P ＜ 0.05).Conclusion HIF-2a could not only affect HUVECs formation but also promote SMMC-7721 cells migration via regulating Ang-2 expression.
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