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目的研究邻苯二甲酸二(2-乙基)己酯(DEHP)与苯并(a)芘(BaP)联合染毒对Chang liver细胞的毒性。方法 DEHP(62.5、125、250、500和1 000μmol/L)与BaP(64μmol/L)单独或联合染毒Chang liver细胞24 h,检测细胞活力、细胞内活性氧(ROS)含量、线粒体膜电位(MMP)、细胞凋亡率和Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤因子2(Bcl-2)以及半胱氨酸蛋白酶-3(caspase-3)蛋白表达量。结果联合染毒各组细胞存活率均低于DEHP单独染毒组(t=6.22、4.64、6.64、5.84、7.29,P<0.01),但胞内ROS水平均升高(t=4.57、2.23、2.39、2.22、2.16,P<0.05或P<0.01);≥250μmol/L DEHP与BaP联合染毒组的MMP分别为(80.12±6.41)%、(69.92±5.56)%和(53.76±1.88)%,均低于BaP单独染毒组的(165.93±5.09)%(t=10.92、12.51、14.62,P<0.01);细胞凋亡率分别为(7.73±1.91)%、(11.00±3.04)%和(14.20±3.96)%,均高于BaP单独染毒组的(1.55±0.21)%(t=3.96、6.06、8.11,P<0.01);此外,活化caspase-3的高表达和升高的Bax/Bcl-2比值也被检出。结论较高浓度DEHP与BaP联合染毒Chang liver细胞促发ROS水平升高和线粒体膜电位降低,并导致了细胞凋亡;Bax、Bcl-2和活化caspase-3参与了此调控过程。
Objective To study the toxicity of DEHP and BaP to Chang liver cells. Methods Chang liver cells were treated with DEHP (62.5, 125, 250, 500 and 1000 micromol / L) and BaP (64 micromol / L) alone or in combination for 24 h. Cell viability, intracellular reactive oxygen species (ROS) (MMP), apoptosis rate, Bcl-2, Bcl-2 and caspase-3 protein expression were detected. Results The survival rate of cells in combination group was lower than that of DEHP group (t = 6.22,4.64,6.64,5.84,7.29, P <0.01), but the intracellular ROS levels were increased (t = 4.57,2.23, (80.12 ± 6.41)%, (69.92 ± 5.56)%, and (53.76 ± 1.88)%, respectively, when treated with ≥250μmol / L DEHP and BaP , Which were lower than those of BaP alone group (165.93 ± 5.09)% (t = 10.92,12.51,14.62, P <0.01). The apoptotic rates were (7.73 ± 1.91)%, (11.00 ± 3.04)% and (14.20 ± 3.96)%, respectively, which were higher than that of BaP alone group (1.55 ± 0.21)% (t = 3.96,6.06,8.11, P <0.01). In addition, the overexpression of activated caspase-3 and the increase of Bax / Bcl-2 ratio was also detected. Conclusions The combination of DEHP and BaP in combination with Chang liver cells induces the increase of ROS level and mitochondrial membrane potential and leads to apoptosis. Bax, Bcl-2 and caspase-3 are involved in this regulation process.