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目的研究抑癌基因p73和生长抑制及DNA损伤诱导基因45α(GADD45α)蛋白对邻苯二甲酸单(2-乙基己基)酯[di(2-ethylhexyl)phthalate,MEHP]染毒人正常肝细胞L02和人肝癌细胞Hep3B周期的影响。方法将处于对数生长期的L02细胞和Hep3B细胞分别暴露于终浓度为0(溶剂对照,DMSO终浓度<1‰)、6.25、12.502、5.00、50.00、100.00μmol/L的MEHP溶液染毒细胞12 h。采用四甲基氮唑蓝(methylthiazoletetrazolium,MTT)比色法检测细胞增值率,采用流式细胞仪检测细胞的周期时相,采用Western blot法检测细胞周期调控蛋白p53、p73和GADD45α的表达量。结果与溶剂对照组相比,仅50μmol/L MEHP染毒L02细胞的增殖率升高(P<0.05),各浓度MEHP染毒Hep3B细胞的增殖率均下降(P<0.01),差异有统计学意义。与溶剂对照组相比,仅100μmol/L MEHP染毒L02细胞的G1期和G2期细胞构成比下降,S期细胞构成比上升,差异均有统计学意义(P<0.05);而100μmol/L MEHP染毒Hep3B细胞的G1期和25.00、50.00、100.00μmol/L MEHP染毒Hep3B细胞的S期细胞构成比下降,50.00和100.00μmol/L MEHP染毒Hep3B细胞的G2期细胞构成比上升,差异均有统计学意义(P<0.01)。与溶剂对照组相比,各浓度MEHP染毒L02细胞均未见p53p、73和GADD45α蛋白表达量明显增加,差异无统计学意义;而各浓度MEHP染毒Hep3B细胞p73蛋白表达量和50.00、100.00μmol/L MEHP染毒Hep3B细胞GADD45α蛋白表达量均升高,差异有统计学意义(P<0.05,P<0.01)。结论一定浓度MEHP(50μmol/L和100μmol/L)可能通过独立于p53途径诱导p73和GADD45α蛋白表达增加而导致p53基因缺失的Hep3B细胞阻滞在G2期。
Objective To investigate the effect of the tumor suppressor gene p73 and growth inhibitory and DNA damage induced gene 45α (GADD45α) on human normal hepatocytes exposed to di (2-ethylhexyl) phthalate (MEHP) L02 and human hepatocellular carcinoma cells Hep3B cycle. Methods L02 cells and Hep3B cells in logarithmic growth phase were exposed to MEHP solution exposed to a final concentration of 0 (solvent control, DMSO final concentration <1 ‰), 6.25,12.502,5.00,50.00,100.00μmol / L 12 h. Cell proliferation rate was measured by methylthiazoletetrazolium (MTT) colorimetric assay. Cell cycle phases were detected by flow cytometry. Western blot was used to detect the expression of p53, p73 and GADD45α. Results Compared with the solvent control group, the proliferation rate of L02 cells treated with 50 μmol / L MEHP alone increased (P <0.05), and the proliferation rate of Hep3B cells treated with MEHP at various concentrations decreased (P <0.01) significance. Compared with the solvent control group, the percentage of cells in G1 phase and G2 phase of L02 cells treated with 100 μmol / L MEHP and the percentage of cells in S phase increased significantly (P <0.05), while 100 μmol / L The cell cycle of S phase in MEHP-exposed Hep3B cells and Hep3B cells in 25.00,50.00,100.00μmol / L MEHP-treated groups decreased, and the percentage of cells in G2 phase of MEHP-exposed Hep3B cells increased by 50.00 and 100.00μmol / L MEOO All were statistically significant (P <0.01). Compared with the solvent control group, no expression of p53p, 73 and GADD45α protein was found in MEHP-treated L02 cells at all concentrations, but the difference was not statistically significant. However, the expression of p73 protein in MEHP-treated Hep3B cells was significantly increased The expression of GADD45α protein in Hep3B cells exposed to μmol / L MEHP was significantly increased (P <0.05, P <0.01). Conclusion A certain concentration of MEHP (50μmol / L and 100μmol / L) may arrest Hep3B cells with p53 gene deletion in G2 phase by inducing p73 and GADD45α protein expression independently of p53 pathway.