摘要:
为探讨邻苯二甲酸二(2-乙基己基)酯(di-(2-ethylhexyl) phthalate, DEHP)及其代谢产物邻苯二甲酸单-2-乙基己酯(mono-(2-ethylhexyl) phthalate, MEHP)对H295R细胞类固醇激素合成关键基因表达的影响，本实验将H295R细胞分别暴露于DEHP(0、1、10、100、1 000 µmol L-1)和MEHP(0、1、10、100、1 000 µmol L-1) 24 h，用MTT法检测细胞活性，并应用荧光定量PCR法分析细胞类固醇激素合成过程中关键酶的基因表达水平。结果显示，1 000 μmol·L-1 DEHP和MEHP对H295R细胞染毒24 h显著降低H295R细胞活力，所以本研究采用了较低的染毒浓度(0、1、10和100 μmol·L-1)对H295R细胞染毒24 h来评估DEHP和MEHP对H295R细胞类固醇激素合成通路的影响。1、10和100 μmol·L-1 DEHP显著增加醛固酮合成酶CYP11B2的基因表达水平。10 μmol·L-1 DEHP显著上调了3-β羟基类固醇脱氢酶(3β-HSD2)的基因表达水平。1、10和100 μmol·L-1 MEHP显著下调了3-β羟基类固醇脱氢酶(3β-HSD1和3β-HSD2)、17β羟基类固醇脱氢酶17β-HSD4、17α羟化酶/17,20裂解酶CYP17和芳香酶CYP19a的基因表达水平。10和100 μmol·L-1 MEHP染毒H295R 24 h显著下调了CYP21和STAR的基因表达水平，然而，10和100 μmol·L-1 MEHP显著上调了CYP11B2的基因表达水平。100 μmol·L-1 MEHP显著下调了17β-HSD1的基因表达水平。上述研究结果表明，DEHP、MEHP都可不同程度影响H295R细胞类固醇激素合成过程中关键基因的表达。MEHP可以通过抑制STAR基因的表达，从而将影响胆固醇在细胞内的转运；并能显著性抑制类固醇激素合成过程中CYP17、CYP19a、3β-HSD1、3β-HSD2、17β-HSD1、17β-HSD4、CYP21基因的表达，最终将抑制H295R细胞中类固醇激素的合成。与DEHP相比，MEHP对H295R细胞类固醇激素合成关键基因表达的影响较明显。
Abstract:
To study the effects of exposure to di(2-ethylhexyl) phthalate (DEHP) and its main metabolite mono (2-ethylhexyl) phthalate (MEHP) on the expression of steroidogenic genes in the H295R cells, H295R cells were exposed to DEHP (0, 1, 10, 100, 1 000 µmol L-1) and MEHP (0, 1, 10, 100, 1 000 µmol L-1) respectively for 24 h, cell viability was assessed by MTT assay, and real time quantitative PCR method was conducted to explore the mRNA expression levels of the key enzymes involved in steroid hormone synthesis. The results showed that 1 000 µmol L-1 DEHP or MEHP significantly reduced cell viability of H295R cells. Therefore, the lower concentrations of DEHP and MEHP were selected to evaluate the effects of DEHP and MEHP on the steroid synthesis pathway of H295R cells in the present study. The expression of aldosterone synthase (CYP11B2) was upregulated significantly upon DEHP at different dose compared to that of the control. Moreover, the transcript level of 3-beta hydroxy steroid dehydrogenase (3β-HSD2) was also increased significantly during exposure to 10 μmol·L-1 DEHP. In contrast, The mRNA levels of 3β-HSD1, 3β-HSD2, 17β-hydroxy steroid dehydrogenase (17β-HSD4), P450 17 alpha-hydroxylase/C17-20-lyase gene (Cyp17) and aromatic enzyme (CYP19a) were significantly decreased in H295R cells after exposure to 1, 10 and 100 μmol·L-1 MEHP. Exposure to MEHP of 10 and 100 μmol·L-1 for 24 h significantly down-regulated the mRNA levels of CYP21 and STAR and up-regulated the expression of CYP11B2 in H295R cells. 100 μmol·L-1 MEHP significantly down-regulated the expression levels of 17β-HSD1. Thus, DEHP and MEHP can affect the expression of key genes involved in steroid synthesis in H295R cells to a certain extent. MEHP can inhibit the expression of STAR gene and affect the transport of steroid hormone in cells, then significantly inhibit the expression of CYP17, CYP19a, 3β-HSD1, 3β-HSD2, 17β-HSD1, 17β-HSD4, CYP21, which were all involved in steroid hormone biosynthesis, and ultimately inhibit the synthesis of steroid hormones in H295R cells. The effects of MEHP on the expression of steroidogenic genes in the H295R cells were more obvious than that of DEHP.
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