DEHP对HepG2细胞糖脂代谢关键基因干扰效应研究

Interference of DEHP on the key genes of glucose and lipid metabolism in HepG2 cells

目的观察邻苯二甲酸二(2-乙基己)酯(DEHP)刺激人肝癌细胞系HepG2细胞后对细胞糖代谢和脂代谢关键基因表达的影响,探讨DEHP对离体培养HepG2细胞糖脂代谢的毒性。方法取对数生长期HepG2细胞分为DEHP染毒组和对照组,染毒组分别以终浓度为5、10、50、100、500和1 000μmol/L的DEHP染毒,对照组分别予相同终浓度的二甲基亚砜处理。培养24 h后收集细胞,采用实时荧光定量聚合酶链式反应,检测DEHP染毒成功的内源性标志物过氧化物酶体增殖物激活受体α(PPARα),糖代谢关键基因葡萄糖-6-磷酸酶(G-6-Pase)和磷酸烯醇式丙酮酸羧激酶(PEPCK),以及脂代谢关键基因硬脂酰辅酶A去饱和酶1(SCD1)、脂肪酸合成酶、固醇调节原件结合蛋白-1c和乙酰辅酶A羧化酶1的mRNA转录水平。校正检验水准为0.008。结果与自身对照组比较,除了5μmol/L剂量外,其余5个剂量DEHP染毒组HepG2细胞PPARαmRNA转录水平均上调(P<0.008),说明DEHP染毒模型诱导成功。与自身对照组比较,100和500μmol/L DEHP染毒组HepG2细胞的G-6-Pase mRNA转录水平均上调(P≤0.008);6个剂量下,2组HepG2细胞的PEPCK mRNA转录水平分别比较,差异均无统计学意义(P>0.008)。除100μmol/L DEHP染毒组SCD1 mRNA转录水平较对照组下调(P<0.008)外,其余剂量下,2组HepG2细胞的4种脂代谢关键基因的mRNA转录水平分别比较,差异均无统计学意义(P>0.008)。结论 DEHP对糖代谢影响主要体现为促进糖异生限速酶G-6-Pase表达的上调。DEHP对HepG2细胞脂代谢的影响较为有限。

Objective To investigate the effects of di-（ 2-ethylhexyl） phthalate（ DEHP） on the expression of the key genes involved in glucose and lipid metabolism,and explore the toxicity of DEHP on the glucose and lipid metabolism in HepG2 cells cultured in vitro. Methods HepG2 cells in logarithmic growth phase were divided into DEHP exposure group and control group. The exposure group was exposed to DEHP with different final concentrations（ 5,10,50,100,500 and1 000 μmol / L）,and the control group was exposed to dimethyl sulfoxide of corresponding concentrations. After 24 hours of DEHP exposure,real-time fluorescence quantitative polymerase chain reaction（ Q-PCR） was applied to detect the level of mRNA transcription of peroxisome proliferators-activated receptor α（ PPARα）, which is an endogenous marker indicating the success of DEHP exposure. In addition,the level of mRNA transcription of key genes involved in glucose and lipid metabolism were also measured by Q-PCR,including glucose-6-phosphatase（ G-6-Pase）,phosphoenolpyruvate carboxykinase（ PEPCK）,stearoyl-coenzyme A desaturase 1（ SCD1）,fatty acid synthase,sterol regulatory elementbinding protein 1c and acetyl Co A carboxylase 1. P ≤0. 008 was considered as statistical significance. Results After DEHP exposure,the mRNA transcription level of PPARα was significantly elevated in all exposure groups（ P 〈0. 008）except for 5 μmol / L DEHP exposure group,which indicated the successful establishment of DEHP exposure model. The mRNA transcription level of G-6-Pase was significantly increased in 100 and 500 μmol / L DEHP exposure groups（ P ≤0. 008） when compared with the controls; the PEPCK mRNA transcription level of showed no significant differences between the 6 DEHP exposure groups and their corresponding control groups（ P 〉0. 008）. The mRNA transcription level of SCD1 was significantly down-regulated in 100 μmol / L DEHP exposure group（ P 〈0. 008） when compared with its control. The mRNA transcription level of other key genes involved in the lipid metabolism were not significantly altered after DEHP exposure（ P 〉0. 008）. Conclusion The effect of DEHP on glucose metabolism was mainly manifested by promoting G-6-Pase gene expression,which is the rate-limiting enzyme for gluconeogenesis. The effect of DEHP on the lipid metabolism of HepG2 cells was limited.