缺氧诱导因子-2α通过血管生成素-2途径调控缺氧诱导血管形成的研究

Hypoxia inducible factor-2 alpha regulating hypoxia-induced angiogenesis via angiopoietin-2 pathway

    摘要
  • 摘要: 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Ang2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±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。

     

    Abstract: 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 rh-Ang-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) Enzymelinked 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 ±s, repeated measurement data were analyzed by the repeated measures ANOVA, comparison among groups and pairwise comparison were conducted respectively by the oneway 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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