NF-κB参与枸橼酸铁铵过载诱导的人肝细胞hepcidin基因表达  

NF-κB participates in hepcidin up-regulation induced by iron overload in HH4 hepatocytes

作　　者：李士伟[1,2] 李想[3] 关锋[1,2] 

机构地区：[1]江南大学糖化学与生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122 [3]江南大学无锡医学院,江苏无锡214122

出　　处：《中国病理生理杂志》2015年第4期695-701,共7页Chinese Journal of Pathophysiology

摘　　要：目的:研究核因子κB(NF-κB)在枸橼酸铁铵过载诱导人肝细胞铁调素(hepcidin)基因表达中的调控作用。方法:依据前期不同浓度枸橼酸铁铵抑制人肝细胞HH4增殖实验结果,选取0.1、1、5和10 mmol/L枸橼酸铁铵处理人肝细胞HH4 48 h,半定量PCR法检测胞内hepcidin的表达水平;利用染色质免疫共沉淀(Ch IP)、电泳迁移率实验(EMSA)和双萤光素酶报告基因检测系统,并结合胞内NF-κB活性抑制实验,确定NF-κB对人hepcidin转录活性的影响。结果:5 mmol/L和10 mmol/L浓度的枸橼酸铁铵处理细胞后,hepcidin表达水平显著增强;Ch IP和EMSA实验证实NF-κB能够与hepcidin基因启动子结合;重组萤光素酶报告质粒TOPFlash-p Hepcidin相对萤光素酶活性明显高于对照组;与重组质粒TOPFlash-p Hepcidin相比,突变重组萤光素酶报告质粒TOPFlash-p Hepcidin-mut相对萤光素酶活性显著降低;NF-κB抑制剂BAY 11-7082显著降低了hepcidin基因的表达。结论:NF-κB参与了铁过载诱导的人hepcidin基因表达。这为进一步深入研究肝细胞铁过载模式下多因子协同调控hepcidin基因表达的具体分子机理奠定了基础。AIM：To study the effects of nuclear factor kappa B （ NF-κB） on human hepcidin expression in fer-ric ammonium citrate （ FAC）-induced HH4 hepatocytes.METHODS：Non-transformed HH4 cells were exposed to FAC at concentrations of 0.1, 1, 5 and 10 mmol/L for 48 h.The expression of iron regulatory gene hepcidin was determined by semi-quantitative RT-PCR.The effects of NF-κB on hepcidin transcriptional activity were detected using chromatin immuno-precipitation （ChIP）, electrophoretic mobility shift assay （EMSA） and dual-luciferase reporter assay system, combined with the inhibition experiments of intracellular NF-κB activity.RESULTS： FAC at concentrations of 5 mmol/L and 10 mmol/L significantly enhanced the expression of hepcidin.The results of ChIP and EMSA showed the binding of NF-κB to the upstream of hepcidin promoter.Treatment with NF-κB inhibitor BAY 11-7082 attenuated hepcidin expression.The lucif-erase activity in the cells transfected with recombinant luciferase reporter plasmid was obviously higher than that in control group.CONCLUSION：NF-κB is the transcription factor that contributes to hepcidin expression in iron overload-induced HH4 cells.

关 键 词：核因子ΚB 染色质免疫共沉淀 电泳迁移率实验 双萤光素酶报告基因检测系统 铁调素 

分 类 号：R363[医药卫生—病理学]