乙肝病毒X基因诱导肝细胞脂肪变性作用机制的研究

摘要] 目的 探讨沉默肝X受体α(liver x receptor alpha，LXRα)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）、shLXRα转染组（转染LXRα质粒）。构建针对LXRα基因的shLXRα质粒，转染HepG2.2.15细胞，荧光显微镜及Western blot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，根据结果予油酸钠刺激细胞，甘油三酯（TG）检测细胞脂肪变程度，RT-PCR检测固醇调节元件结合蛋白(sterol regulatory element binding protein-1c，SREBP-1c)mRNA的表达，Western blot检测乙肝病毒x蛋白(hepatitis B virus x protein, HBx)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蛋白表达明显下降，于转染后48～72h表达最低（0.43±0.03） vs (0.61±0.03)，(0.33±0.03) vs (0.69±0.02)，P＜0.01]，差异有统计学意义；随着油酸钠处理时间延长各组TG含量、SREBP-1c mRNA水平、HBx和FAS蛋白表达均逐渐增加，同一时间点，HBx蛋白各组无明显差异（P>0.05），而TG含量、SREBP-1c mRNA水平、FAS蛋白与空白对照组和阴性对照组比较， shLXRα转染组中表达较低TG：（21.21±3.39）μg/mg vs（32.61±5.09）μg/mg]；SREBP-1c：（0.418±0.051 vs 0.516±0.037；FAS：0.48±0.03 vs 0.63±0.03，P<0.01），差异有统计学意义。结论 HBx对脂代谢的调控是通过LXRα/ SREBP-1c /FAS途径实现的。

Mechanism on hepatitis B virus X gene-induced hepatic steatosis

Objective To investigate the effect of liver X receptor α(LXRα) gene silencing on lipid metabolism-related genes in HepG2.2.15 cells.Methods HepG2.2.15 cells were divided into blank control group(without transfection),negative control group(transfected with HK plasmid),and shLXRα group(transfected with shLXRα plasmid).The shLXRα plasmids carrying LXRα gene were constructed and were used to transfect HepG2.2.15 cells using PolyJetTM reagent.Green fluorescent protein and LXRα protein expression were examined by fluorescence microscope and Western blotting analysis 24-96 h after transfection,so as to identify the best interference time.Then cells were treated with agonist T0901317 for 24 h or 48 h;and the content of triglyceride(TG) was observed to detect the degree of steatosis by biochemical assay.The expression of sterol regulatory element binding protein-1c(SREBP-1c) mRNA was detected by RT-PCR and the expression of hepatitis B virus X(HBx) protein and fatty acid synthase(FAS) protein was tested by Western blotting analysis.Results The shLXRα plasmid was constructed and transfected into HepG2.2.15 cells successfully.Compared with blank and negative control groups,LXRα protein was markedly decreased in the shLXRα group,with the lowest level found at 48-72 h after transfection(P<0.01).After cells were stimulated with T0901317,HBx and FAS protein expression,the content of TG,and SREBP-1c mRNA expression gradually increased with the prolongation of stimulation period,and there was no significant difference in HBx expression at the same time point between different groups.FAS protein,TG contents,and SREBP-1c mRNA in shLXRα group were significantly lower than those in the other two groups(P<0.01).Conclusion HBx can regulate lipid metabolism through LXRα/SREBP-1c/FAS pathway.