乙肝病毒X蛋白对油酸钠诱导HepG2细胞脂质沉积的影响

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。

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

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 pIRES2-EGFP-HBx was transiently transfected into HepG2 cells to establish HepG2-HBx cell model for HBx expression.HepG2 cells transfected with empty vector pIRES2-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 sodium 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 sterol regulatory element binding protein-1(SREBP-1) and liver X receptor alpha(LXRα) and protein levels of HBx,LXRα and fatty acid synthase(FAS),respectively.Results At 16 h after transfection,GFP expression 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 LXRα and SREBP-1 as well as protein levels of LXRα 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-related genes of HBx-LXRα-SREBP-1/FAS pathway,thereby increasing sodium oleate-induced lipid accumulation in HepG2 cells.