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目的探讨沉默肝X受体α(liver X receptorα,LXRα)基因对HepG2.2.15细胞脂质代谢相关基因表达的影响。方法设立空白对照组(不转染任何质粒)、阴性对照组(转染阴性HK质粒)和shLXRα转染组(转染shLXRα质粒)。构建针对LXRα基因的shLXRα质粒并转染HepG2.2.15细胞,用荧光显微镜及蛋白质印迹法检测转染质粒24～96h绿色荧光蛋白和LXRα蛋白的表达以确定质粒的最佳干扰时间,根据结果给予激动剂T0901317刺激细胞,用三酰甘油(TG)含量检测肝细胞脂肪变性程度,RT-PCR检测固醇调节元件结合蛋白1c(sterol regulatory element binding protein-1c,SREBP-1c)mRNA的表达,蛋白质印迹法检测乙肝病毒X(hepatitis B virus X,HBx)蛋白及脂肪酸合成酶(fatty acid synthase,FAS)蛋白的表达。结果成功构建shLXRα质粒并转染HepG2.2.15细胞。与空白对照组和阴性对照组比较,shLXRα转染组LXRα蛋白表达下降,于转染后48～72h表达最低,差异有统计学意义(P<0.01);随着激动剂T0901317处理时间延长,各组HBx和FAS蛋白表达、TG含量、SREBP-1cmRNA水平均逐渐增加,同一时间点HBx蛋白在各组差异无统计学意义(P>0.05),而FAS蛋白、TG含量、SREBP-1cmRNA水平,在shLXRα转染组中的表达较空白对照组和阴性对照组低,差异有统计学意义(P<0.01)。结论 HBx对脂代谢的调控可能部分通过LXRα/SREBP-1c/FAS途径实现。
Objective To investigate the effect of liver X receptor α (LXRα) gene on lipid metabolism related gene expression in HepG2.2.15 cells. Methods The blank control group (no plasmid transfection), negative control group (transfected negative HK plasmid) and shLXRα transfection group (transfected with shLXRα plasmid) were established. The shLXRα plasmid targeting LXRα gene was constructed and transfected into HepG2.2.15 cells. The expression of green fluorescence protein (GFP) and LXRα protein in transfected plasmids were detected by fluorescence microscopy and Western blotting to determine the best interference time of plasmids. The results were stimulated The expression of sterol regulatory element binding protein-1c (SREBP-1c) mRNA was detected by RT-PCR, and Western blotting was used to detect the degree of fatty degeneration of hepatocytes by using T0901317 as stimulator. Method to detect the expression of hepatitis B virus X (HBx) protein and fatty acid synthase (FAS) protein. Results The shLXRα plasmid was successfully constructed and transfected into HepG2.2.15 cells. Compared with the blank control group and the negative control group, the expression of LXRαprotein in shLXRαtransfected group decreased at 48-72h after transfection, the difference was statistically significant (P <0.01); with the prolongation of agonist T0901317 HBx and FAS protein expression, TG content, SREBP-1cmRNA levels were gradually increased at the same time point HBx protein in each group was no significant difference (P> 0.05), while FAS protein, TG content, SREBP-1cmRNA levels in The expression of shLXRα in transfection group was lower than that in blank control group and negative control group (P <0.01). Conclusion The regulation of lipid metabolism by HBx may be partly through LXRα / SREBP-1c / FAS pathway.