邻苯二甲酸二（2-乙基己）酯对原代培养的人早孕绒毛细胞滋养细胞凋亡的影响

Effects of di-（ 2-ethylexyl ） phthalate on apoptosis of cytotrophoblasts in early pregnancy

目的 探讨邻苯二甲酸二（2-乙基己）酯（DEHP）对原代培养的人早孕绒毛细胞滋养细胞凋亡及凋亡相关基因Bcl-2和bax表达的影响.方法 用浓度为0、25、50、100 μmol/L的DEHP作用于原代培养的人早孕绒毛细胞滋养细胞24 h,逆转录（RT）PCR法检测滋养细胞凋亡相关基因Bcl-2和bax的mRNA表达水平;蛋白印迹法检测Bcl-2和bax的蛋白表达水平;原位末端脱氧核苷酸转移酶标记（TUNEL）法和双染流式细胞术检测细胞滋养细胞凋亡情况.结果 （1）Bcl-2表达水平：当DEHP浓度为0、25、50、100 μmol/L时,Bcl-2 mRNA表达水平分别为1.00±0.05、1.03±0.04、1.04±0.03、1.04±±0.04,分别比较,差异均无统计学意义（P〉0.05）;Bcl-2蛋白表达水平分别为0.11±0.02、0.11±0.04、0.12±0.02、0.12±0.03,分别比较,差异也无统计学意义（P〉0.05）.（2）bax表达水平：当DEHP浓度为50、100 μmol/L时,bax mRNA表达水平分别为0.96±0.04、1.02±0.04,与DEHP浓度为0 μmol/L时（0.81±0.05）比较,差异有统计学意义（P〈0.05）,bax蛋白表达水平分别为0.63±0.04、0.81±0.04,与DEHP浓度为0 μmol/L时（0.23±0.05）比较,差异也有统计学意义（P〈0.05）.（3）细胞凋亡率：浓度为50、100 μmol/L的DEHP作用24 h后,双染流式细胞术检测细胞滋养细胞凋亡率分别为（18.8±2.6）%和（20.3±2.0）%,与DEHP浓度为0 μmol/L时[（10.6±1.4）%]比较,差异有统计学意义（P〈0.05）;TUNEL法检测细胞滋养细胞凋亡率为（18.1±4.6）%和（19.5±1.2）%,与DEHP浓度为0 μmol/L时[（11.2±3.1）%]比较,差异也有统计学意义（P〈0.05）.结论 DEHP可通过增加细胞凋亡相关基因bax的表达,促进细胞滋养细胞凋亡,但对Bcl-2的表达无明显影响.

Objective To investigate the influence of di-（2-ethylexyl） phthalate （DEHP） on cell apoptosis and expression of Bcl-2 and bax in cultured human first trimester cytotrophoblasts. Methods Human first trimester cytotrophoblasts were cultured with DEHP at concentration of 0, 25, 50, 100 μmol/Lfor 24 hours. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling （TUNEL） and flow cytometer method. The expression of apoptosis-associated genes, including Bcl-2 and bax, were detected by reverse transcription （RT）-PCR in cultured cytotrophoblast cells. The protein expression of Bcl-2 and bax in cytotrophoblast cells was measured by western blot. Results （1） The expression of Bcl-2： when incubated with DEHP at concentration of 0, 25,50 and 100 μmol/L, the expression of Bcl-2 were 1.00 ± 0.05, 1.03 ± 0.04, 1.04 ± 0.03, 1.04± 0.04,which did not show statistical difference （ P 〉 0.05 ）. The expression of Bcl-2 protein were 0.11 ± 0.02,0.11 ±0.04, 0.12±0.02, 0.12 ±0.03, which also didn＇t reach statistical difference （P〉0.05）. （2）The expression of bax： when incubated with DEHP at concentration of 50 and 100 μmol/L, the expression of bax protein were 0.63 ± 0.04 and 0.81 ± 0.04, which were significantly higher than 0.23 ± 0.05 with DEHP at 0 μmol/L （P 〈 0.05）. The expression of bax mRNA were 0.96 ± 0.04 and 1.02 ± 0.04, which was significantly higher than 0.81 ±0.05 with DEHP at 0 μmol/L （P 〈 0.05）. （3） Apoptosis： when incubated with DEHP at concentration of 50 and 100 μmol/L for 24 hours, the apoptotic cell ratio were （ 18.8 ± 2.6） % and （ 20.3 ± 2.0） % by annexin V-FITC/PI staining, which were significantly higher than （10.6±1.4）% at 0 μmol/L and （18.1 ±4.6）% and （19.5 ±1.2）% by TUNEL staining, which were significantly higher than （ 11.2 ± 3.1 ） % at 0 μmol/L of DEHP （P 〈 0.05）. Conclusion DEHP could induce apoptosis of cytotrophoblast cells by increasing bax gene expression, but had no effect on Bcl-2 expression.