CAJ | 학술논문

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)α20ng/ml]、高脂[低密度脂蛋白(LDL) 100 μ g/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达情况.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SREBP2shRNA-HepG2.在NC shRNA-HepG2细胞中,TNF α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TNF α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.
목적 탐토침묵담고순조절원건결합단백(SREBP)2대염증인자소치HepG2세포내지질이상적취적영향.방법 통과지질체전염SREBP2-shRNA질립、G418항성사선HepG2세포,건립침묵SREBP2적HepG2은정세포주(SREBP2 shRNA-HepG2)급음성대조질립HepG2은정세포주(NC shRNA-HepG2).대량충세포분별급여무혈청배양처리(대조조)、염증인자[종류배사인자(TNF)α20ng/ml]、고지[저밀도지단백(LDL) 100 μ g/ml]화연합간예(20ng/ml TNFα +100μg/ml LDL)처리.유홍O염색급매법검측세포중지질적취정황,실시정량PCR화Western blot 분별검측SREBP2급기하유파기인저밀도지단백수체(LDLr)화3-간-3-갑기무이선보매A(HMGCoA)환원매적기인화단백표체정황.대수거용단인소방차분석급2×2×2석인설계방차분석비교. 결과 성공건립음성대조은정세포주NC shRNA-HepG2급억제SREBP2표체적은정세포주SREBP2shRNA-HepG2.재NC shRNA-HepG2세포중,TNF α처리사세포내담고순적취증가,병능상조SREBP2하유파기인LDLr、HMGCoA환원매기인화단백적표체,기mRNA상대표체량분별위1.68±0.03、1.31±0.05,F치분별위107.42,59.08,P치균＜0.01 ;기단백상대표체량분별위1.49±0.10、1.54±0.06,F치분별위46.24,247.10,P치균＜0.01.이재SREBP2 shRNA-HepG2세포중,TNFα대포내담고순침적적작용가이피명현감약,진일보기인화단백검측결과현시,염증인자TNF α대LDLr、HMGCoA환원매적상조작용역피명현억제. 결론 염증인자통과촉진SREBP2급기하유파기인표체치HepG2세포내담고순이상적취,이통과RNA간우억제SREBP2적표체,가이명현감경염증인자소치적세포내담고순적이상적취.
Objective To investigate the effect of RNA interference (RNAi)-mediated silencing of the SREBP2 on inflammatory cytokine-induced cholesterol accumulation in HepG2 cells.Methods Shorthairpin (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 enzymatic 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 shRNA HepG2 and NC shRNA 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 gene 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.Conclusion 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.