天津医药

天津医药 ›› 2017, Vol. 45 ›› Issue (3): 225-228.doi: 10.11958/20160773

• 细胞与分子生物学 •    下一篇

缺氧环境下雷公藤甲素对人脐静脉内皮细胞HIF1α和VEGF基因表达的影响

田园， 张苗苗， 时文涛， 李申， 王玉川， 何玉坤， 李卫东△   

  1. 天津医科大学基础医学院遗传学系
  • 收稿日期:2016-08-05 修回日期:2017-01-23 出版日期:2017-03-15 发布日期:2017-03-21
  • 通讯作者: 李卫东 E-mail:tianyuan2015@tmu.edu.cn
  • 基金资助:
    国家自然科学基金资助项目 （81070576）； 天津市自然科学基金重点项目 （12JCZDJC24700）

The effect of triptolide on HIF1α and VEGF expressions under hypoxia in human umbilical vein endothelial cells

TIAN Yuan, ZHANG Miao-miao, SHI Wen-tao, LI Shen, WANG Yu-chuan, HE Yu-kun, LI Wei-dong△   

  1. Department of Genetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China
  • Received:2016-08-05 Revised:2017-01-23 Published:2017-03-15 Online:2017-03-21
  • Contact: LI Wei-dong E-mail:tianyuan2015@tmu.edu.cn

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摘要： 摘要： 目的 探讨缺氧环境下雷公藤甲素 （TP） 对人脐静脉内皮细胞 （HUVECs） 中缺氧诱导因子 1α （HIF1α） 和血管内皮生长因子 （VEGF） 表达的影响。方法 （1） HUVECs 加入 0、 40、 80、 160、 320 nmol/L 的 TP （分别为缺氧组， TP40组、 TP80 组、 TP160 组、 TP320 组） 在缺氧条件下 （37 ℃、 5%CO2、 1%O2、 94%N2） 培养 12 h， 同时设常氧组 （不加 TP） 作为 对照， Western blot 检测各组 HIF1α 蛋白表达。（2）细胞设 TP80 组、 缺氧组和常氧组， 免疫荧光法检测 HIF1α 在细胞中的表达定位。（3） 设 TP80 组和缺氧组， 处理结束后 Western blot 检测 VEGF 蛋白表达水平。（4） 设缺氧组、 TP80 组，TP80+KF20 组（80 nmol/L TP 和 20 μmol/L KC7F2）、 TP80+KF30 组（80 nmol/L TP 和 30 μmol/L KC7F2）， 处理 12 h 后Western blot 检测各组 HIF1α、 VEGF 蛋白表达。结果 （1）常氧条件下 HUVECs 不表达 HIF1α； 缺氧条件下， TP80组 HIF1α 表达水平较缺氧组明显升高（P < 0.05）， TP160 组和 TP320 组 HIF1α 表达水平与缺氧组相比无明显变化。（2）细胞免疫荧光结果显示 HIF1α 主要表达于细胞核。（3） TP80 组 VEGF 表达水平较缺氧组升高（P < 0.05）。（4）经KC7F2 干预后， TP80+KF20 组和 TP80+KF30 组 HIF1α、 VEGF 表达水平较 TP80 组明显降低 （P < 0.05）。结论 缺氧环境下 TP 可通过提高 HIF1α 和 VEGF 表达来促进内皮细胞的增殖。

关键词: 雷公藤甲素, 内皮细胞, 缺氧诱导因子 1, α 亚基, 血管内皮生长因子类, 人脐静脉内皮细胞, KC7F2, 细胞增殖

Abstract: Abstract: Objective To investigate the effect of triptolide (TP) on the expression of hypoxia inducible factor 1 alpha(HIF1α) and vascular endothelial growth factor (VEGF) in the human umbilical vein endothelial cells (HUVECs) under hypoxia. Methods (1) HUVECs were treated with 0, 40, 80, 160 and 320 nmol/L TP (named with hypoxia group, TP40 group, TP80 group, TP160 group and TP320 group, respectively) under the hypoxic condition (37 ℃, 5% CO2, 1% O2, 94%N2) for culturing 12 hours. Meanwhile, cells cultured under normoxia condition (without TP added) were set as the normoxia group. Western blot assay was used to detect the expression of HIF1α in each group. (2) The cells were divided into normal control group, hypoxia group and TP80 group. The immunofluorescence method was performed to detect the localization of HIF1α in cells. (3) Expressions of VEGF were detected by Western blot assay in TP80 group and hypoxia group. (4) The cells were divided into hypoxia group, TP80 group, TP80 +KF20 group (80 nmol/L TP and 20 μmol/L KC7F2), and TP80+KF30 group (80 nmol/L TP and 30 μmol/L KC7F2). After 12- hour culturing, Western blot assay was used to detect the expressions of HIF1α and VEGF in each group. Results (1) Under the normoxia condition, no HIF1α was detected in HUVECs. The expression level of HIF1α was significantly increased in TP80 group than that in hypoxia group (P < 0.05),while there was no significant change in expression of hypoxia HIF1α in TP160 group and TP320 group compared with that of hypoxic group. (2) The immunofluorescence result showed that HIF1α was mainly expressed in the nucleus. (3) The expression of VEGF was significantly increased in TP80 group than that in hypoxia group (P < 0.05). (4) After theintervention of KC7F2, HIF1α and VEGF expression levels were significantly decreased in the TP80+KF20 group and the TP80+ KF30 group than those in the TP80 group (P < 0.05). Conclusion TP can improve the expression of HIF1α and VEGF to accelerate the proliferation of endothelial cells under hypoxia condition.

Key words: triptolide, endothelial cells, hypoxia-inducible factor 1, alpha subunit, vascular endothelial growth factors, human umbilical vein endothelial cells, KC7F2, cell proliferation