缺氧应激对HepG-2增殖活性和HIF-1α、MMP-2表达的影响

EFFECTS OF HYPOXIA STRESS ON THE PROLIFERATION OF HEPG-2 CELL AND EXPRESSIONS OF MMP-2 AND HIF-1α

目的研究缺氧应激对人肝癌细胞HepG-2增殖和缺氧诱导因子-1α(HIF-1α)、基质金属蛋白-2(MMP-2)表达的影响。方法将缺氧(5%O2、5%CO2、90%N2)和常氧环境中培养的HepG-2细胞,在相差显微镜下观察细胞形态变化;用四甲基偶氮唑蓝(MTT)比色法测定细胞增殖活性;用逆转录聚合酶链反应(RT-PCR)法测定常氧组和缺氧4、12、24h组HepG-2细胞HIF-1αmRNA和MMP-2mRNA的表达量;用双抗体夹心酶联免疫吸附实验(ELISA)法测定常氧和缺氧12、24h组细胞培养上清MMP-2蛋白含量。结果同时相缺氧组HepG-2细胞增殖活性与常氧组比较,差异有统计学意义(P<0.05)。常氧下HepG-2细胞不表达HIF-1αmRNA,缺氧4h时HIF-1αmRNA表达量最高,随后下降(P<0.01),但仍高于常氧组;缺氧4h时MMP-2mRNA表达量最高,随后下降,但仍高于常氧组(P<0.01);缺氧组HIF-1αmRNA和MMP-2mRNA呈高度正相关关系。缺氧24h组HepG-2细胞培养上清液中的MMP-2蛋白含量与常氧24h组含量比较,差异有统计学意义(P<0.05)。结论缺氧应激(5%O2)促进人肝癌细胞HepG-2的增殖作用并上调HIF-1α和MMP-2的表达量。

Objective To investigate whether hypoxia stress enhanced the proliferation of human hepatoeellular carcinoma cell HepG -2 and upregulated the expressions of matrix metalloproteinase -2 （MMP -2 ） and hypoxia - inducible factor - 1 α（ HIF - 1 α ） Methods HepG- 2 cell was euhured in hypoxia（5% O2, 5% CO2, 90% N2 ） and normoxia incubator. The morpological changes of HepG - 2 cells were studied microscopically anti the proliferatinn of HepG -2 were detected by methyl thiazolyl tetrazolium （MTT）. The expressions of HIF - 1α and MMP 2 mRNA in cuhured ItepG -2 were determined by reverse transcription polymerase chain reaction（ RT- PCR） The MMP- 2 protein level in supernatants was monitored by enzyme- linked immunoabsorbent assay （ELISA）. Results There was a little morphological change of HepG - 2 cells in normoxia and hypoxia group, a great change of cell proliferation in corresponding time （ P 〈 0.05 ）. The expressions of MMP - 2 mRNA and HIF - 1 α mRNA were dramatically enhanced in 4 - hour hypoxia group ; then decreased in 12 - hour and 24 - hour hypoxia group （ P 〈 0.01 ）, but still higher than those in normoxia group. There is a significantly positive correlation between the expressions of MMP - 2 mRNA and HIF - 1 α mRNA in hypoxia group . The expression of MMP - 2 protein in 24 - hour hypoxia group was significantly higher than that in 24 -hour normoxia group（ P 〈 0.05 ）. Conclusion Hypoxia stress（5% O2 ） enhanced the proliferation of HepG -2 cell and upregulated expressions of MMP- 2 and HIF -1α.