DEHP对HTR-8/Svneo细胞功能的影响及机制研究

Effect and Mechanism of DEHP on the Function of HTR-8/Svneo Cells

目的:通过研究常见的邻苯二甲酸酯类中邻苯二甲酸二(2-乙基己)酯(DEHP)对人滋养细胞HTR-8/SVneo(简称HTR-8)功能的影响及机制,初步探讨DEHP导致稽留流产的机制。方法:采用不同浓度DEHP溶液干预HTR-8细胞,CCK-8检测细胞毒性、流式细胞仪检测细胞凋亡及细胞周期的变化。用Western-blot方法探讨DEHP对凋亡相关蛋白的影响,分析DEHP致凋亡的可能通路。结果:(1)CCK-8检测细胞毒性示:DEHP≥30 ng/ml作用24小时及48小时,HTR-8细胞活性明显受抑制(P<0.05);DEHP浓度为20 ng/ml时作用24小时,对HTR-8细胞活性无明显抑制作用;但作用48小时,HTR-8细胞活性同样受到抑制(P<0.01)。(2)凋亡实验结果:小剂量DEHP(15 ng/ml)能抑制HTR-8细胞凋亡(P<0.05);而大剂量DEHP(30 ng/ml)促进细胞凋亡(P<0.05)。(3)流式细胞检测发现:DEHP浓度15 ng/ml作用HTR-8细胞24小时,细胞阻滞于S期(P<0.05);DEHP浓度30 ng/ml作用HTR-8细胞24小时,细胞阻滞于G0/G1期(P<0.01)。(4)DEHP浓度15 ng/ml作用HTR-8细胞6小时,促凋亡因子Bax蛋白表达下降而凋亡抑制因子Bcl-2的蛋白表达增加。结论:大剂量DEHP(≥30 ng/ml)使滋养细胞活力明显受抑制,且不受作用时间的影响。小剂量DEHP(≤15 ng/ml)对滋养细胞活力无明显影响,但抑制其凋亡,可能与小剂量的DEHP下调Bax、上调Bcl-2的表达有关,这可能是DEHP致稽留流产的机制之一。

Objective：To explore the probable effect of DEHP（one of most common phthalate esters）on the function of human trophoblastic cell line HTR-8/SVneo（HTR-8） and discuss the mechanisms of DEHP leading to missed abortion. Methods. To observe changes in cell toxicity with different concentrations of DEHP by CCK-8,and detect apoptosis and cell cycle by flow cytometry. To investigate the effect of DEHP on apoptosis associated protein by Western-blot method,and the possible pathway of DEHP inducing apoptosis. Results① CCK-8 assay for cytotoxicity showed that HTR-8 cells activity was significantly inhibited（ P 〈 0.05）by DEHP ≥ 30 ng/ml for 24 h and 48 h and 20 ng/ml for48 h,while the activity of HTR-8 cells was not inhibited（P〉0.05） by 20 ng/ml for 24 h. ②Cell apoptosis： small dose of DEHP（15 ng/ml）inhibited the apoptosis of HTR-8 cells （ P 〈 0.05）, while large dose of DEH P （ 30 ng/ml） promoted cell apoptosis （ P 〈 0.05 ）. ③ FCM results showed that HTR-8 cell cycle arrested in S phase（ P〈0.05）by 15 ng/ml of DEHP for24 hand stayed in G0/G1 phase （P〈0.01）by 30 ng/ml of DEHP for 24 h. ④Decreased expression of Bax（apoptosis inducer）and increased expression of Bcl-2 （apoptosis inhibitor）were observed in HTR-8 cells with 15ng/ml DEHP for 6 h. Conclu- sions.Large dose of DEHP （≥ 30 ng/ml）inhibits activity of trophoblast significantly with no effect from time. Small dose of DEHP（15 ng/ml）has no obvious effect on trophoblast cell viability, but inhibits the apoptosis of cell. Down-regulation of Bax and up-regulation of Bcl-2 by small dose of DEHP may be one of the mechanisms of missed abortion induced by DEHP.