| 摘要: |
| 目的 探讨沉默肝X受体α (liver X receptor α, 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24～96 h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48～72 h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-1c mRNA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-1c mRNA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。 |
| 关键词: 肝X受体α 乙肝病毒X基因 脂代谢障碍 RNA干扰 |
| DOI：10.3724/SP.J.1008.2012.026 |
| 投稿时间：2011-11-04修订日期：2011-11-29 |
| 基金项目:国家自然科学基金(30871160). |
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| Mechanism on hepatitis B virus X gene-induced hepatic steatosis |
| ZHANG Qin1,PENG Jun2,SHEN Wei1* |
(1. Department of Gastroenterology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China
2. Department of Gynaecology and Obstetrics, First People’s Hospital of Liangshan Prefecture, Xichang 615000, Sichuan, China
*Corresponding author.) |
| Abstract: |
| Objective To investigate the effect of liver X receptor α (LXRα) gene silencing on lipid metabolism-related genes in HepG2.2.15 cells. MethodsHepG2.2.15 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. ResultsThe 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). ConclusionHBx can regulate lipid metabolism through LXRα/SREBP-1c/FAS pathway. |
| Key words: liver X receptor α hepatitis B virus X gene lipid metabolism disorders RNA interference |
