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目的:研究RNAi技术沉默载脂蛋白C-Ⅲ基因(ApoC3)表达后对HepG2细胞内外脂质含量及其他脂代谢相关基因表达的影响。方法:合成3对针对ApoC3mRNA不同位点序列的小干扰RNA(siRNA),并分组转染至人肝细胞株HepG2,实时荧光定量PCR和酶联免疫吸附法检测ApoC3mRNA及蛋白质的表达水平,筛选出沉默效果最佳的一对siRNA。将最有效的siRNA转染HepG2细胞,48h后测定细胞内外脂质含量以及脂代谢相关基因的mRNA表达水平。结果:25nmol/L siRNA和3μl/孔(24孔板)转染试剂的转染效率最高。ApoC3-siRNA-3对ApoC3基因的沉默效果最好,对mRNA的抑制率为94.6%,上清液中检测不到ApoC-Ⅲ。ApoC3-siRNA-3沉默ApoC3基因后细胞内三酰甘油(TG)和胆固醇含量显著高于对照组,细胞外TG含量显著低于对照组。与对照组比较,ApoC3-siRNA-3沉默组载脂蛋白B100基因mRNA表达水平显著增强,肝脂酶基因mRNA表达水平显著降低。结论:ApoC3基因沉默可显著升高HepG2细胞内TG含量及显著降低细胞外TG含量,其可能机制是通过减少极低密度脂蛋白(VLDL)颗粒的分泌以及增加VLDL残粒的摄取。
OBJECTIVE: To study the effect of silencing the expression of apolipoprotein C-Ⅲ gene (ApoC3) by RNAi in vitro and in vivo on the lipid profile and other lipid metabolism related genes in HepG2 cells. Methods: Three small interfering RNAs (siRNAs) targeting different ApoC3 mRNA loci were synthesized and transfected into HepG2 cells. The expression of ApoC3 mRNA and protein was detected by real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay Silence best pair of siRNA. HepG2 cells were transfected with the most effective siRNA, and the content of lipid in the cells and the mRNA expression of lipid metabolism related genes were determined 48h later. Results: The transfection efficiency of 25nmol / L siRNA and 3μl / well (24-well plate) transfection reagent was the highest. ApoC3-siRNA-3 had the best silencing effect on ApoC3 gene, with an inhibitory rate of 94.6% on mRNA and no ApoC-Ⅲ in supernatant. ApoC3-siRNA-3 silencing ApoC3 gene intracellular triglyceride (TG) and cholesterol content was significantly higher than the control group, the extracellular TG content was significantly lower than the control group. Compared with the control group, the ApoC3-siRNA-3 silencing group significantly increased the mRNA expression of apolipoprotein B100 gene and the hepatic lipase gene mRNA expression level significantly decreased. Conclusion: ApoC3 gene silencing can significantly increase the content of TG in HepG2 cells and decrease the content of extracellular TG significantly. The mechanism may be through decreasing the secretion of VLDL particles and increasing the uptake of VLDL remnants.