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目的采用油酸诱导Hep G2细胞发生脂变,建立脂肪变性细胞模型,观察SIRT1/UCP2通路在脂肪变性Hep G2细胞能量代谢中的调控机制。方法采用含有2 mmol/L油酸的DMEM培养基诱导Hep G2细胞24 h后,建立脂肪变性Hep G2细胞模型,并设置对照组比较。以油红O染色观察细胞内脂滴形成状况,并用全自动生化仪检测细胞上清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)含量和细胞内甘油三酯(TG)含量;采用流式细胞仪测定线粒体膜电位的改变,采用磷钼酸比色试剂盒测定细胞内三磷酸腺苷(ATP)含量,运用Western印迹法检测各组细胞SIRT1和UCP2蛋白的表达。结果与对照组比较,模型组细胞内橘红色脂滴大量形成,且TG含量明显升高(P<0.01),线粒体膜电位及细胞内ATP含量显著降低(P<0.05,P<0.01),SIRT1蛋白表达均显著降低(P<0.05),UCP2蛋白表达显著升高(P<0.01)。结论油酸诱导的脂肪变性Hep G2细胞模型可以出现脂质代谢紊乱和线粒体能量代谢失衡,其机制可能与细胞内SIRT1/UCP2通路的激活有关。
OBJECTIVE: To study the regulation of SIRT1 / UCP2 pathway in the energy metabolism of Hep G2 cells induced by oleic acid and to establish the model of steatosis. Methods Hep G2 cells were induced by DMEM containing 2 mmol / L oleic acid for 24 h, then the Hep G2 cell model was established and compared with the control group. The formation of intracellular lipid droplets was observed by oil red O staining. The contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and intracellular triglyceride TG). The changes of mitochondrial membrane potential were measured by flow cytometry. The ATP content in cells was determined by phosphomolybdate colorimetric kit. The expression of SIRT1 and UCP2 protein in each group was detected by Western blotting. Results Compared with the control group, the lipid droplets in the model group were formed in large numbers and the content of TG was significantly increased (P <0.01), and the mitochondrial membrane potential and intracellular ATP content were significantly decreased (P <0.05, P <0.01) (P <0.05), UCP2 protein expression was significantly increased (P <0.01). Conclusion Oleic acid-induced hepatic steatosis model of Hep G2 cells may show dyslipidemia and mitochondrial energy metabolism imbalance, which may be related to the activation of intracellular SIRT1 / UCP2 pathway.