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目的探讨肿瘤坏死因子(TNF)-α对人肝癌细胞HepG2肿瘤抑制基因RECK表达的影响,检测核转录因子(NF)-kB在RECK基因表达的调控中的作用。方法将培养的细胞分为3组,A组用4种浓度的TNF-α(1、5、50、100μg/L)作用人肝癌细胞HepG2细胞株6h,B组用浓度为50μg/L的TNF-α分别作用HepG2细胞1、6、12、24 h,逆转录-聚合酶链反应(RT-PCR)检测RECKmRNA的表达；C组将NF-kB抑制剂二硫代氨基甲酸吡硌烷(PDTC,10mol/L)与TNF-α(50λg/L)同时作用以及TNF-α(50μg/L)单独作用于HepG2细胞6 h,凝胶滞留电泳实验(EMSA)DNA结合反应检测NF-kB的活性,RT-PCR和Western blot分别检测RECK基因mRNA和蛋白的表达,明胶酶谱试验检测细胞MMP-9的活性。结果HepG2细胞中RECK基因无表达,TNF-α作用后RECK的表达显著增高,作用呈时间和剂量依赖关系(P＜0．01),TNF-α能激活NF-kB并能显著抑制HepG2细胞MMP-9的活性(P＜0．01),而PDTC能显著的抑制这种作用(P＜0．01)。结论TNF-α能上调RECK基因在肝癌细胞系HepG2中的表达同时抑制细胞MMP-9的活性。而NF-kB可能参与了这一过程。
Objective To investigate the effect of tumor necrosis factor-α (TNF-α) on the expression of RECK in human hepatoma HepG2 tumor suppressor gene and to investigate the role of nuclear factor-kappaB (NF-κB) in the regulation of RECK gene expression. Methods The cultured cells were divided into 3 groups: group A was treated with 4 concentrations of TNF-α (1,5,50,100μg / L) for 6 hours, and group B was treated with 50μg / L of TNF The expression of RECK mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) in HepG2 cells at 1, 6, 12 and 24 hours respectively. In group C, the NF-kB inhibitor pyrrolidine dithiocarbamate , 10mol / L) combined with TNF-α (50λg / L) and TNF-α (50μg / L) alone for 6 h. EMSA DNA binding assay was used to detect the activity of NF- The mRNA and protein expression of RECK were detected by RT-PCR and Western blot, respectively. Gelatin zymography was used to detect the activity of MMP-9. Results RECK gene was not expressed in HepG2 cells, and the expression of RECK was significantly increased after TNF-α treatment (P <0.01). TNF-α could activate NF-κB and significantly inhibit the expression of MMP in HepG2 cells -9 (P <0.01), while PDTC significantly inhibited this effect (P <0.01). Conclusion TNF-α can up-regulate the expression of RECK gene in HepG2 cell line and inhibit the activity of MMP-9. NF-kB may be involved in this process.