摘要
目的探讨缺氧诱导因子-2α(hypoxia-inducible factors-2α,HIF-2α)表达水平对人肝癌细胞株HepG2增殖能力的影响。方法用氯化钴(CoCl_2)诱导细胞模拟缺氧微环境,并根据CoCl_2浓度不同分为50μmol/L、100μmol/L、200μmol/L、400μmol/L组,选择CoCl_2浓度为200μmol/L模拟肿瘤内缺氧微环境。采用siRNA干扰沉默HepG2细胞中HIF-2α的表达,分别应用RT-PCR、Western blotting方法检测HIF-2αmRNA和蛋白的表达,MTT方法检测HepG2细胞增殖,绘制细胞生长曲线,流式细胞技术观察细胞周期的改变。结果建立细胞缺氧模型,作为低氧组;采用浓度为50 nmol/L的HIF-2αsiRNA转染缺氧环境下的HepG2细胞,作为低氧+HIF-2αsiRNA组,结果显示:低氧组、低氧+Control siRNA组的HIF-2αmRNA和蛋白表达显著高于常氧组和低氧+HIF-2αsiRNA组(P<0.05);细胞生长曲线结果显示:低氧组、低氧+Control siRNA组的光密度值显著高于常氧组和低氧+HIF-2αsiRNA组(P<0.05);细胞周期结果显示:低氧组、低氧+Control siRNA组HepG2细胞在DNA合成前期(G0/G1期)细胞比率显著低于常氧组和低氧+HIF-2αsiRNA组,合成期(S)及合成后期(G2/M)细胞比率显著高于常氧组和低氧+HIF-2αsiRNA组(P<0.05)。结论缺氧微环境通过促进人肝癌细胞株HepG2细胞中HIF-2α的表达进一步促进HepG2细胞增殖,而针对HIF-2α的靶向siRNA能有效抑制肝癌HepG2细胞中HIF-2α的表达,从而抑制HepG2细胞的增殖。
Objective To investigate the impacts of expression level of hypoxia-inducible factors-2α （HIF-2α） on the multiplication capacity of human hepatocellular carcinoma cell HepG2. Methods Cobalt chloride （ CoC12 ） cells were induced by simulated hypoxic mieroenvironment, and depending on the concentration of CoC12 into 50 μmol/L group, 100 μmol/L group, 200 μmol/L group, 400 μmol/L group, 200 μmol/L concentration of CoC12 was selected to simulate hypoxia in tumor mieroenvironment, siRNA was transfected into human hepatoeellular carcinoma HepG2 cell to silent HIF-2α. Application of RT-PCR and Western blotting method respectively to detect the expression of HIF-2α mRNA and protein, MTT method to detect the proliferation of HepG2 cells, to curve the cell growth, and Flow eytometry to observe the changes of cell cycle. Results Establishment of cellular hypoxia model as the hypoxia group. The con- centration of 50 nmol/L HIF-2α siRNA transfeetion of HepG2 cells under hypoxia environment as the hypoxia ＋ HIF-2α siRNA group. Results showed that the HIF-2α mRNA and protein expression of hypoxia group and hypoxia ＋ control siR- NA group were significantly higher than normal oxygen group and hypoxia ＋ HIF-2α siRNA group （P 〈 0.05 ）. The results of cell growth curve showed that the optical density of hypoxia group and hypoxia ＋ control siRNA group were sig- nificantly higher than normal oxygen group and hypoxia ＋ HIF-2α siRNA group （P 〈 0.05）. Cell cycle showed that in the beginning of DNA synthesis in HepG2 cells （ G0/G1 period） cells of hypoxia group and hypoxia ＋ control siRNA group were significantly lower than normal oxygen group and hypoxia ＋ HIF-2α siRNA group; but synthesis phase （S） and later synthesis （G2/M） cell rate were significantly higher than normal oxygen group and hypoxia ＋ HIF-2α siRNA group （P 〈 0.05）. Conclusion Hypoxic mieroenvironment can promote the expression of HIF-2α in HepG2 cells to further promote the proliferation of HepG2 cells. Whereas, for siRNA targeting HIF-2α can effectively inhibit the expr-ession of hepatocellular carcinoma HepG2 cells in the HIF-2α, thus inhibit the proliferation of HepG2 cells.
出处
《胃肠病学和肝病学杂志》
CAS
2017年第8期921-925,共5页
Chinese Journal of Gastroenterology and Hepatology
关键词
缺氧诱导因子-2Α
氯化钴
RNA干扰
肝癌
细胞增殖
Hypoxia induced factors-2α
COC12
RNA interference
Hepatocellular carcinoma
Multiplication
作者简介
李伟伟，硕士，主治医师，研究方向：肝病临床和基础。E-mail：liweiweicyk@163．com
通讯作者：宋新文，硕士，主任医师，研究方向：肝病临床和基础。E-mail：283521725@qq．com
