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目的分析在α-亚麻酸(ALA,C18:3)干预后,胰岛素抵抗Hep G2(insulin resistance Hep G2,IR-Hep G2)细胞模型脂类合成关键基因表达水平的变化。方法 Hep G2细胞造模期分为两组,由无血清培养基培养的对照组(normal control group,NC)以及含有0.25 mmol/L软脂酸的无血清培养基培养的软脂酸组(palmitic acid,PA),培养24 h后鉴定细胞胰岛素敏感性并测定细胞内胆固醇(total cholesterol,TCH)、甘油三酯(Tryglyceride,TG)水平,然后用ALA替代20%的PA培养12 h,再次鉴定细胞胰岛素敏感性并检测细胞内TCH、TG水平,real time q PCR检测TG、TCH合成关键基因mRNA水平,Western blot检测TG、TCH合成关键基因蛋白表达量。结果 IR-Hep G2细胞模型建立成功,并且PA组TCH水平显著高于NC组(P=0.0016),出现了脂代谢紊乱;ALA干预IR-Hep G2细胞12 h后,细胞活性有所恢复,与PA组相比,ALA组胰岛素诱导基因(insulin induced genes,INSIGs)及脂类合成关键蛋白的mRNA表达无明显变化;与基因表达结果不一致的是,ALA干预后可以特异性提升细胞内INSIG-2蛋白相对表达量,但对INSIG-1蛋白相对表达量没有明显影响。同时,ALA可以显著抑制55k Da固醇调节元件结合蛋白-2(sterol regulatory element-binding protein-2,SREBP-2)、HMG-Co A还原酶(3-hydroxy-3-methyl glutaryl coen-zyme A reductase,HMGCR)及脂肪酸合酶(fatty acid synthase,FASN)蛋白的表达,且ALA组细胞内TG水平显著降低(P=0.0119)。结论 0.05 mmol/L ALA干预IR-Hep G2细胞后,通过提升INSIG-2蛋白的表达,抑制SREBP-2从内质网到高尔基体的剪切成熟活化,降低细胞内55k Da SREBP-2水平及HMGCR的表达,并且抑制FASN表达,从而抑制了TG/TCH的合成。
OBJECTIVE: To analyze the changes of key gene expressions of lipid synthesis in insulin resistance Hep G2 (IR-Hep G2) cell model induced by α-linolenic acid (ALA, C18: 3). Methods Hep G2 cells were divided into two groups: normal control group (NC) and palmitic group (0.25 mg / L) and serum-free medium containing palmitic acid, PA). The cell insulin sensitivity was determined 24 h after incubation and the levels of total cholesterol (TCH) and tryglyceride (TG) were determined. The cells were cultured with ALA instead of 20% PA for 12 h and identified again Cell insulin sensitivity and detect intracellular TCH, TG levels, real time q PCR detection of TG, TCH synthesis key gene mRNA levels, Western blot detection TG, TCH synthesis of key gene protein expression. Results The IR-Hep G2 cell model was established successfully and the level of TCH in PA group was significantly higher than that in NC group (P = 0.0016). Lipo-metabolic disturbance was found. After ALA intervention for 12 h, the cell activity recovered, Compared with PA group, there was no significant change in mRNA expression of insulin induced genes (INSIGs) and key proteins of lipid synthesis in ALA group. The results of gene expression were different from that of ALA group Protein relative expression, but had no significant effect on the relative expression of INSIG-1 protein. At the same time, ALA can significantly inhibit the expression of 55k Da sterol regulatory element-binding protein-2 (SREBP-2), HMG-Co A-reductase reductase (HMGCR) and fatty acid synthase (FASN) protein expression in the ALA group were significantly lower than those in the ALA group (P = 0.0119). CONCLUSIONS: 0.05 mmol / L ALA can inhibit the maturation and activation of SREBP-2 from endoplasmic reticulum to the Golgi by increasing the expression of INSIG-2 protein and decrease the level of 55 kDa SREBP-2 in IR-Hep G2 cells HMGCR expression, and inhibit FASN expression, thereby inhibiting the TG / TCH synthesis.