TNF-α对肝癌细胞VEGF和MMP-9表达的影响

Effects of Tumor Necrosis Factor-α on Vascular Endothelial Growth Factor and Matrix Metalloproteinase-9 Expression in Hepatic Cancer Cell Line HepG2

目的研究肿瘤坏死因子α(TNFα)对肝癌HepG2细胞血管内皮生长因子(VEGF)和基质金属蛋白酶9(MMP9)表达的影响。方法不同浓度(10、102、103、104及105U/L)TNFα作用于HepG2细胞不同时间(24、48及72h)后在相差显微镜下观察细胞形态变化;四甲基偶氮唑蓝(MTT)比色法测定细胞增殖活性;双抗体夹心酶联免疫吸附实验(ELISA)测定细胞培养上清液中的VEGF和MMP9蛋白含量;免疫细胞化学方法观察细胞浆中的VEGF和MMP9蛋白表达;逆转录酶聚合酶链反应(RT PCR)半定量法测定HepG2细胞VEGF和MMP9mRNA含量。结果①不同浓度的TNFα作用后,HepG2细胞形态无明显变化,细胞增殖活性比较,差异无显著性意义(P>0.05);随各浓度TNFα作用时间延长,HepG2细胞形态无明显变化,但细胞增殖活性逐渐增高(P<0.05)。②不同浓度的TNFα作用不同时间后,细胞培养上清液中的VEGF和MMP9蛋白含量明显增加(P<0.05),且存在时间依赖性,并在TNFα浓度小于或等于104U/L时存在浓度依赖性;HepG2细胞VEGF和MMP9mRNA表达亦明显升高(P<0.05),其促进作用的变化趋势同VEGF和MMP9蛋白含量结果一致,但不存在时间依赖性。③104U/L的TNFα作用48h后,HepG2细胞浆中的棕色颗粒明显增加。结论TNFα能够在核酸和蛋白水平上促进肝癌细胞合成并分泌VEGF和MMP9,从而间接促进肿瘤血管生成。

Objective To investigate whether tumor necrosis factor-α (TNF-α) enhance the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9(MMP-9) in hepatic cancer cell line HepG2 or not. Methods Cultured HepG2 cells were treated by TNF-α with various concentration and time. The morphological changes of HepG2 cells were studied microscopically and the proliferation of HepG2 were detected by methyl thiazolyl tetrazolium (MTT). The expression of VEGF and MMP-9 mRNA in cultured HepG2 were determined by relative quantitative reverse transcription polymerase chain reaction. The VEGF and MMP-9 protein level in supernatants and in cytoplasm were determined by enzyme-linked immunosorbent assay (ELISA) and by immunocytochemical staining, respectively.Results There was a little morphological changes in HepG2 with TNF-α treatment, but no change of cell proliferation in corresponding time. The expression of VEGF and MMP-9 mRNA was enhanced gradually with the TNF-α concentration increasing, the VEGF and MMP-9 protein level in supernatants and in cytoplasm was elevated gradually with the concentration increasing. There was a dependance on the concentration when the concentration of TNF-α was lower than or equal to 10~4 U/L. Furthermore, the effect of promotion was close to peak when the TNF-α concentration up to 10~4 U/L; but no time-effect pattern observed. Conclusion TNF-α can enhance the expression of VEGF and MMP-9 at the level of mRNA and protein in hepatic cancer cell line.