DEHP及其代谢产物MEHP对MLTC-1细胞凋亡和类固醇激素合成基因表达的影响

Effects of DEHP and its Metabolite MEHP on Cell Apoptosis and the Expression of Steroidogenic Genes in MLTC-1 Cells

目的实验测评DEHP及其主要代谢产物MEHP暴露对MLTC-1细胞类固醇激素合成关键基因表达的影响。方法将MLTC-1分别暴露于DEHP(0,1,10,100,1 000μmol/L)和MEHP(0,1,10,100,1 000μmol/L)24 h,采用Annexin V/PI双染法测定细胞凋亡,并应用荧光定量PCR法分析细胞类固醇激素合成过程中涉及关键酶的基因表达水平。结果凋亡实验结果显示DEHP(100和1 000μmol/L)和MEHP(10和1 000μmol/L)显著诱导MLTC-1细胞晚期凋亡的发生,1 000μmol/L DEHP和MEHP显著降低了MLTC-1活力。所以本研究采用了较低的染毒浓度(1,10和100μmol/L)对MLTC-1染毒24 h来评估DEHP和MEHP对MLTC-1类固醇合成通路的影响。荧光定量结果显示1μmol/L DEHP显著增加MLTC-1 CYP17A1基因表达水平,而100μmol/L DEHP显著抑制MLTC-1 CYP17A1、CYP11A1和SF-1基因表达水平。10μmol/L DEHP显著增加MLTC-1外CYP1A1基因表达水平。100μmol/L DEHP显著抑制MLTC-1黄体激素受体LHR基因表达水平。1μmol/L MEHP显著增加MLTC-1 3β-HSD基因表达水平。然而MEHP对类固醇激素合成过程中一些酶如STAR、CYP17A1、CYP11A1、重要调节因子胰岛素样激素3 INSL-3、SF-1、关键受体AR和LHR基因表达水平均无显著影响。结论 DEHP和MEHP诱导MLTC-1凋亡,DEHP和MEHP都可影响MLTC-1细胞类固醇激素合成过程中关键基因的表达,从而引起内分泌干扰。

Objectives To study the effects of DEHP and its main metabolite mono（ 2-ethylhexyl） phthalate（ MEHP） exposure on steroidogenesis in MLTC-1. Methods MLTC-1 were exposed to DEHP（ 0,1,10,100,1 000 μmol/L） and MEHP（ 0,1,10,100,1 000 μmol/L） for 24 h respectively. Apoptosis was detected by Annexin V/PI double staining,and real time quantitative RT-PCR method was conducted to explore the mRNA expression levels of the key enzymes involved in the synthesis of steroid hormones. Results Apoptosis result showed100 and 1 000 μmol/L DEHP and MEHP significantly induced the late apoptosis of MLTC-1,1 000 μmol/L DEHP and MEHP significantly reduced the cell viability. Therefore,the lower concentrations of DEHP and MEHP were selected to evaluate the effect of DEHP and MEHP on the steroid synthesis pathway of MLTC-1 in the present study. RT-PCR result showed 1 μmol/L DEHP significantly increased the mRNA expression level of CYP17A1,while 100 μmol/L DEHP significantly inhibited mRNA expression level of CYP17A1,CYP11A1,key regulatory factor（ SF-1）. 10 μmol/L DEHP significantly increased the expression level of CYP1 A1. 100 μmol/L DEHP significantly inhibited the expression of LHR. 1 μmol/L MEHP significantly increased the expression of 3β-HSD. However,MEHP had no significant effect on the expression of some enzymes such as STAR,CYP17A1,CYP11A1,insulin-like hormone 3（ INSL-3）,SF-1,AR and LHR. Conclusions DEHP and MEHP promoted MLTC-1 apoptosis and affected the expression of key genes in steroid hormone synthesis of MLTC-1,which may cause endocrine disruption.