乙肝病毒X蛋白对油酸钠诱导HepG2细胞脂质沉积的影响

Effect of hepatitis B virus X protein on sodium oleate-induced lipid accumulation in HepG2 cells

目的探讨乙肝病毒X蛋白(hepatitis B virus X protein,HBx)是否增加油酸钠诱导HepG2细胞的脂质沉积。方法将质粒pIRES2-eGFP-HBx瞬时转染入HepG2细胞中,建立表达HBx的细胞模型(HepG2-HBx);以转染空载体pIRES2-eGFP(HepG2-pIRES2)和HepG2细胞(HepG2)作对照。观察转染后绿色荧光蛋白(GFP)的表达;转染后16 h开始用油酸钠处理各组细胞24、48 h(分别命名为HBx/OA组、空/OA组、G2/OA组),细胞内甘油三酯(TG)含量测定及油红O染色了解细胞内脂质沉积情况;在油酸钠处理细胞24 h,RT-PCR法检测SREBP-1和LXRα的mRNA表达水平,Westernblot检测HBx、LXRα及FAS蛋白表达水平。结果转染后16 h HepG2-HBx细胞和HepG2-pIRES2细胞中开始有GFP的表达,提示转染成功;仅在HepG2-HBx细胞内有HBx表达,表明HepG2-HBx细胞模型构建成功。在相同油酸钠处理的条件下,HBx/OA组细胞内脂质含量和TG含量与对照组相比均明显增加(P<0.01)。在油酸钠处理细胞24 h,HBx/OA组细胞内LXRα、SREBP-1的mRNA表达量和LXRα、FAS蛋白表达量较对照组均明显增加(P<0.01/0.05)。结论 HBx通过上调HBx-LXRα-SREBP1/FAS通路脂质合成相关基因表达,可能增加HepG2-HBx细胞对外界脂代谢紊乱因素的易感性,从而增加油酸钠诱导HepG2细胞脂质沉积。

Objective To investigate whether hepatitis B virus X protein （HBx） increases sodium oleate-induced lipid accumulation in HepG2 cells. Methods HBX gene eukaryotic expression vector plRES2-EGFP-HBx was transiently transfected into HepG2 cells to establish HepG2-HBx cell model for HBx expression. HepG2 cells transfected with empty vector plRES2-EGFP （HepG2-pIRES2） and untransfected HepG2 cells （HepG2） were used as controls. Expression of green fluorescent protein （GFP） was observed by fluorescence microscope after transfection. At 16 h after transfection, each group of cells was treated with sodi- um oleate for 24 and 48 h （HBx/OA group, empty/OA group and G2/OA group, respectively）. Lipid accumulation in each group of cells was observed by triglyceride （TG） content detection and oil red O staining. At 24 h after sodium oleate treatment, RT-PCR and Western blot were applied to measure mRNA levels of stero] regulatory element binding protein-1 （ SREBP-1 ） and liver X receptor alpha （ LXRct ） and protein levels of HBx, LXRct and fatty acid synthase （FAS）, respectively. Results At 16 h after transfection, GFP expres- sion was detected in HepG2-HBx and HepG2-pIRES2 cells, while HBx expression was only detected in HepG2- HBx cells. These suggested that HepG2-HBx cell model for HBx expression was successfully obtained. Compared with those of the control groups, TG and lipid contents were significantly increased in HBx/OA group （P 〈 0.01 ） after the cells treated with sodium oleate. At 24 h after sodium oleate treatment, mRNA levels of LXRot and SREBP-1 as well as protein levels of LXRct and FAS in the HBx/OA group were significantly higher than those in the control groups （ P 〈 0.01/0.05 ）. Conclusion HBx increases the susceptibility of HepG2- HBx cells to factors causing lipid metabolism disorder through up-regulating expressions of lipid synthesis-relatedgenes of HBx-LXRct-SREBP-1/FAS pathway, thereby increasing sodium oleate-induced lipid accumulation in HepG2 cells.