沉默胆固醇调节原件结合蛋白2基因对炎症因子所致HepG2细胞内胆固醇异常积聚的影响  

Effect of RNAi-mediated silencing of SREBP2 gene on inflammatory cytokine-induced cholesterol accumulation in HepG2 cells

作　　者：廖梭蕾[1] 赵蕾[1] 陈曜[1] 李青[1] 陈昱杨[1] 阮雄中[1] 陈压西[1] 

机构地区：[1]重庆医科大学脂糖代谢性疾病重庆市重点实验室（脂质研究中心）,400016

出　　处：《中华肝脏病杂志》2012年第7期526-531,共6页Chinese Journal of Hepatology

基　　金：国家自然科学基金（30871159、81070631、81070317、81030008）;重庆市教委科学技术研究项目（KJ100320）

摘　　要：目的探讨沉默胆固醇调节原件结合蛋白（SREBP）2对炎症因子所致HepG2细胞内脂质异常积聚的影响。方法通过脂质体转染SREBP2-shRNA质粒、G418抗性筛选HepG2细胞，建立沉默SREBP2的HepG2稳定细胞株（SREBP2 shRNA-HepG2）及阴性对照质粒HepG2稳定细胞株（NC shRNA—HepG2）。对两种细胞分别给予无血清培养处理（对照组）、炎症因子[肿瘤坏死因子（TNF）α 20ng／ml]、高脂[低密度脂蛋白（LDL）100μg／ml]和联合干预（20ng／ml TNFα＋100μg／ml LDL）处理。油红O染色及酶法检测细胞中脂质积聚情况，实时定量PCR和Western blot分别检测SREBP2及其下游靶基因低密度脂蛋白受体（LDLr）和3-羟-3-甲基戊二酰辅酶A（HMGCoA）还原酶的基因和蛋白表迭隋况。对数据用单因素方差分析及2×2×2析因设计方差分析比较。结果成功建立阴性对照稳定细胞株NC shRNA—HepG2及抑制SREBP2表达的稳定细胞株sREBP2 shRNA—HepG2。在NC shRNA—HepG2细胞中，TNFα处理使细胞内胆固醇积聚增加，并能上调SREBP2下游靶基因LDLr、HMGCoA还原酶基因和蛋白的表达，其mRNA相对表达量分别为1．68±0．03、1．31±0．05，F值分别为107．42，59．08，P值均〈0．01；其蛋白相对表达量分别为1．49±0．10、1．54±0．06，F值分别为46．24，247．10，P值均〈0．01。而在SREBP2 shRNA—HepG2细胞中，TNFα对胞内胆固醇沉积的作用可以被明显减弱，进一步基因和蛋白检测结果显示，炎症因子TNFα对LDLr、HMGCoA还原酶的上调作用亦被明显抑制。结论炎症因子通过促进SREBP2及其下游靶基因表达致HepG2细胞内胆固醇异常积聚，而通过RNA干扰抑制SREBP2的表达，可以明显减轻炎症因子所致的细胞内胆固醇的异常积聚。Objective To investigate the effect of RNA interference （RNAi）-mediated silencing of the SREBP2 on inflammatory cytokine-induced cholesterol accumulation in HepG2 cells. Methods Short- hairpin （sh）RNA targeting SREBP2 or negative control （NC） shRNA were transfected into HepG2 cells by a liposomal method. G418-selective culturing was used to obtain the SREBP2 shRNA HepG2 and NC shRNA HepG2 cell lines. The two cell lines were cultured in serum-free medium and left untreated （control） or treated with TNF-α （20 ng/ml）, low-density lipoprotein （LDL） loading （100 μg/ml）, or a combination LDL plus TNF-α treatment. Lipid accumulation was evaluated by oil red O （ORO） staining. Intracellular cholesterol level was measured by enzyrnatie assay. The mRNA and protein levels of SREBP2 and its downstream target genes, LDL receptor （LDLr）, and HMGCoA reductase, were measured by real-time PCR and Western blotting, respectively. Results SREBP2 shR.NA HepG2 and NC sb_RNA HepG2 stable cell lines were successfully established. ORO staining and cholesterol quantitative analysis showed that LDL loading significantly increased intracellular cholesterol and that expression of SREBP2 further exacerbated the inflammatory cytokine-induced lipid accumulation, as seen in NC shRNA HepG2 cells. LDL loading ofNC shRNA HepG2 decreased the gone and protein expressions of SREBP2, LDLr, and HMGCoA reductase, but the suppressive effect was overridden by inflammatory cytokine. SREBP2 shRNA HepG2 cells showed lower levels of cholesterol accumulation under LDL loading and inflammatory stress conditions. Moreover, the mRNA and protein levels of SREBP2, LDLr, and HMGCoA reductase were much lower than in NC shRNA HepG2 cells under the same conditions. Conelusion Inflammatory cytokine exacerbated cholesterol accumulation in HepG2 via disrupting SREBP2. RNAi-mediated inhibition of SREBP2 expression significantly ameliorated the cholesterol accumulation induced by inflammatory cytokine.

关 键 词：脂肪肝 胆固醇 肿瘤坏死因子 胆固醇调节原件结合蛋白2 

分 类 号：R735.7[医药卫生—肿瘤]