邻苯二甲酸二(2-乙基已基)酯对人神经母细胞瘤细胞增殖的影响

Growth-promoting effect of di-2-ethylhexyl phthalate on human neuroblastoma cells

目的探讨环境内分泌干扰物邻苯二甲酸二(2-乙基已基)酯(di-2-ethylhexyl phthalate,DEHP)对人神经母细胞瘤细胞增殖的影响及机制。方法体外培养人神经母细胞瘤SK-N-SH细胞,分为5组:不加干预药物(对照组)、加17β-雌二醇(17β-estradiol,E2组)、加DEHP(DEHP组)、同时加E2和磷脂酰肌醇3-激酶(phosphatidylinositol-3-kinases,PI3K)抑制剂LY294002(E2+LY294002组)、同时加DEHP和LY294002(DEHP+LY294002组)。检测各组细胞0、2、5d时光吸收度值(absorbance value,AV),5d时DNA增殖指数(proliferation index,PI),凋亡指数(apoptotic index,AI)、Capase-3蛋白、蛋白质丝氨酸苏氨酸激酶(protein-serine-threonine kinase,Akt)以及磷酸化Akt(phosphor-AktSer473,p-AktSer473)蛋白表达。结果E2和DEHP组2、5d时AV值及5d时PI值均较对照组明显增高(P<0.01),而E2+LY294002和DEHP+LY294002组则较E2和DEHP组明显降低(P<0.01),各组AI及Caspase-3蛋白表达无明显变化。5d时p-AktSer473蛋白表达,E2和DEHP组较对照组增加(P<0.01),而E2+LY294002和DEHP+LY294002组则较E2和DEHP组明显减少(P<0.01),各组Akt蛋白表达无明显变化。结论DEHP可促进人神经母细胞瘤SK-N-SH细胞体外增殖,作用与雌激素相仿。该促增殖效应可能通过PI3K/Akt信号通路起作用,与细胞凋亡途径无关。

Objective To investigate the effect of di-2-ethylhexyl phthalate （DEHP） on the proliferation of SK-N-SH human neuroblastoma ceils and its underlying mechanism. Methods Cells were cultured in estrogen-free improved Dulbecco＇s Modified Eagle＇s Medium and then divided into 5 groups： no treatment （control group）; treated with 17β-estradiol （E2 group）; treated with DEHP （DEHP group）; treated with both E2 and phosphatidylinositol-3-kinases （PI3K） inhibitor LY294002 （E2 ＋ LY294002 group）; treated with both DEHP and LY294002 （DEHP ＋ LY294002 group）. The absorbance value （AV） was measured on day 0, 2, and 5. DNA proliferation index （PI） and apoptotic index （AI） were determined by flow cytometry on day 5. Caspase-3 protein, protein-serine-threonine kinase （Akt） and phosphor-Akt （Ser473） protein expression were analyzed by Western blot on day 5. Results The AV of All groups increased on day 2, and 5. The AV of E2 and DEHP groups were higher than that of the control group （P〈0.001）, but the AV of E2 ＋ LY294002 and DEHP＋ LY294002 groups were lower than those of E2 and DEHP groups （P〈0. 01 ） on day 2 and 5. On day 5, PI of E2 and DEHP groups were also higher than that of control （P〈0. 01 ）. However, PI of E2 ＋ LY294002 and DEHP ＋ LY294002 groups were lower than those of E2 and DEHP group （P〈0.01） on day 5. There was no significant difference in AI and caspase 3 protein expression among the groups. At the same time, phosphor-Akt （Ser473） protein expression of E;2 and DEHP groups increased obviously, compared with the control group. Compared with E, and DEHP groups, E2＋ LY294002 and DEHP ＋ LY294002 groups decreased significantly. However, Akt protein expression was equal among those groups. Conclusions DEHP can promote the growth of SK-N-SH cells to a level similar to that of E2, with activation of the PI3K/Akt signaling pathway.