胰岛素对人单核/巨噬细胞ACAT1基因P1和P7启动子活性的影响  

Effect of insulin on transcriptional activity of human ACAT1 gene P1 and P7 promoters in THP-1 cells and THP-1-derived macrophages

作　　者：葛晶[1] 成蓓[1] 何平[1] 戚本玲[1] 柯丽[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院老年病科,湖北武汉430022

出　　处：《中国病理生理杂志》2012年第11期1955-1960,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30471921)

摘　　要：目的:研究胰岛素对人单核/巨噬细胞酰基辅酶A:胆固醇酰基转移酶1(acyl coenzyme A:choles-terol acyltransferase 1,ACAT1)基因P1和P7启动子的活性及其转录产物表达的影响,从而探讨它们在胰岛素调控ACAT1基因表达中的作用。方法:将人ACAT1基因P1和P7启动子调控的报告基因载体pGL3E-P1和pGL3E-P7瞬时转染入体外培养人THP-1单核细胞系,给予不同剂量的胰岛素处理,通过双萤光素酶报告系统检测P1和P7启动子的活性。体外培养THP-1细胞和由佛波脂诱导分化的巨噬细胞,同样用不同剂量的胰岛素干预24 h,应用逆转录聚合酶链反应(RT-PCR)检测THP-1细胞ACAT1基因P1和P7启动子转录产物表达,用SYBR GreenⅠ实时定量RT-PCR方法检测THP-1细胞和诱导分化的巨噬细胞ACAT1 mRNA的表达。结果:不同剂量胰岛素处理的人THP-1细胞,与对照组相比,ACAT1基因P1启动子的活性及其转录产物量均显著增强,并随胰岛素剂量的增加而升高。应用实时定量RT-PCR方法检测ACAT1 mRNA的表达与上述结果相一致。结论:胰岛素可通过激活人单核/巨噬细胞ACAT1基因P1启动子上调ACAT1 mRNA的表达,其作用呈剂量依赖性。AIM： To investigate the effect of insulin on the transcriptional activtity of acyl coenzyme A： cholesterol acyltransferase 1（ACAT1） gene P1 and P7 promoters in THP-1 cells and THP-1-derived macrophages.METHODS： The human monocytic leukemia cell line THP-1 was chosen in the study.The differentiation of THP-1 cells into macrophages was induced by stimulating with phorbol myristate acetate（PMA） for 48 h.The luciferase reporter vector containing P1 or P7 promoter of ACAT1 gene was transfected into THP-1 cells by DEAE-dextran method.The luciferase activity was determined by dual-luciferase reporter assay system.Total RNA was extracted with TRIzol from THP-1 cells and THP-1-derived macrophages exposed to different concentrations of insulin for 24 h.The P1 transcript and P7 transcript of ACAT1 gene were detected by RT-PCR in THP-1 cells,and real-time RT-PCR with SYBR green I was used to quantitate the mRNA expression of ACAT1 in THP-1 cells and THP-1-derived macrophages.RESULTS： The transcriptional activity of ACAT1 gene P1 promoter and the relative expression level of ACAT1 gene P1 transcript significantly increased in a dose-dependent manner in insulin-treated THP-1 cells compared with the untreated cells,and no effect of insulin on P7 promoter was observed.Consistent with these results,real-time RT-PCR also showed that the expression level of ACAT1 mRNA was significantly up-regulated in a dose-dependent manner in insulin-treated cells in comparison with the untreated THP-1 cells and THP-1-derived macrophages.CONCLUSION： Insulin up-regulates ACAT1 gene expression by activating P1 promoter in a dose-dependent manner.

关 键 词：胰岛素 酰基辅酶A胆固醇酰基转移酶1 动脉粥样硬化 启动子 转录活性 

分 类 号：R543.5[医药卫生—心血管疾病]