脂联素对HepG2细胞中脂肪酸合成酶及激素敏感性脂肪酶基因启动子活性的影响  

Effects of adiponectin on the gene promoter activities of fatty acid synthase and hormone sensitive lipase in HepG2 cells

作　　者：许瀚元 朱惠娟 潘慧 阳洪波 王林杰 龚凤英 XU Han-yuan;ZHU Hui-juan;PAN Hui;YANG Hong-bo;WANG Lin-jie;GONG Feng-ying(Department of Endocrinology, Key Laboratory of Endocrinology of National Health Commission, Peking Union Medical College Hospital, CAMS & PUMC, Beijing 100730, China)

机构地区：[1]中国医学科学院,北京协和医学院,北京协和医院内分泌科,国家卫生健康委员会内分泌重点实验室,北京100730

出　　处：《基础医学与临床》2021年第1期13-19,共7页Basic and Clinical Medicine

基　　金：国家自然科学基金(81370898,30771026,30540036);北京市自然科学基金(7182130,7082079);人社部留学人员科技活动项目择优资助经费(201401)(启动类);高等学校博士学科点专项科研基金(20040023030);国家临床重点专科建设项目(WBYZ2011-873);协和中青年科研基金(2013-020)。

摘　　要：目的构建含人脂肪酸合成酶(FAS)及激素敏感性脂肪酶(HSL)基因启动子的荧光素酶报告基因表达质粒,探究脂联素对人肝癌细胞系HepG2中FAS及HSL基因启动子活性的影响。方法扩增人FAS及HSL启动子序列-622~+3 bp片段与-697~+53 bp片段,构建含人FAS及HSL启动子的pGL3-hFAS625-Luc(hFAS 625-Luc)与pGL3-hHSL750-Luc(hHSL 750-Luc)荧光素酶报告基因表达质粒。脂质体瞬时转染HepG2细胞,观察0.5~10.0μg/mL脂联素作用24 h或者5μg/mL脂联素作用2~32 h细胞中荧光素酶活性的变化。结果hFAS 625-Luc与hHSL 750-Luc荧光素酶表达质粒均能在HepG2细胞中良好表达。1.0~10.0μg/mL脂联素作用24 h能剂量依赖性地促进转染hFAS 625-Luc及hHSL 750-Luc质粒的HepG2细胞中荧光素酶的表达,最高分别达到对照组的1.76倍(P<0.01)和1.37倍(P<0.01)。5μg/mL脂联素作用于转染hFAS 625-Luc质粒的HepG2细胞4 h和8 h能够促进荧光素酶的表达(P<0.01),作用16 h和32 h则抑制荧光素酶的表达(P<0.01);而脂联素对转染hHSL 750-Luc质粒HepG2细胞作用不同时间(2~32 h)均能促进荧光素酶的表达,最高为对照组的1.37倍(P<0.01)。结论成功构建含人FAS与HSL启动子荧光素酶报告基因质粒。脂联素能够剂量依赖性地促进HepG2细胞中FAS及HSL启动子活性。脂联素作对FAS启动子的活性影响与作用时间有关,而对HSL启动子活性的影响一直表现为促进作用。Objective To investigate the effects of adiponectin on human FAS and HSL gene promoter activities in human hepatocarcinoma cell line HepG2 transfected with plasmids containing human FAS or HSL promoters fused to a luciferase reporter gene.Methods The luciferase reporter gene expression plasmids pGL3-hFAS625-Luc(hFAS625-Luc)and pGL3-hHSL750-Luc(hHSL 750-Luc)were constructed.HepG2 cells were then transfected with these plasmids by lipofectamine method.The FAS and HSL promoter activities was evaluated by testing the luciferase activity through incubation with 0.5-10.0μg/mL adiponectin)for 24 h or 5μg/mL adiponectin for 2-32 h.Results Both hFAS 625-Luc and hHSL 750-Luc plasmids were well expressed in HepG2 cells.1.0-10.0μg/mL adiponectin could progressively increase the luciferase expression in HepG2 cells transfected with hFAS 625-Luc and hHSL 750-Luc plasmids with the maximum effect of 1.76 folds(P<0.01)and 1.37 folds of the controls,respectively,at the concentration of 10.0μg/mL(P<0.01).Moreover,5μg/mL adiponectin promoted the expression of luciferase in HepG2 cells transfected with hFAS 625-Luc plasmids at 4 h and 8 h(P<0.01),and inhibited the expression of luciferase at 16 h and 32 h(P<0.01).Adiponectin promoted the expression of luciferase in HepG2 cells transfected with HSL 750-Luc within different durations(2~32 h),and the highest was 1.37 times of the control group at 2 h(P<0.01).Conclusions The luciferase reporter gene plasmids containing human FAS and HSL promoters are successfully constructed.Adiponectin promotes FAS and HSL promoter activities in HepG2 cells with a dose-dependent manner.Adiponectin has a double effect on FAS promoter activity in different times,while it has a promoting effect on HSL promoter activity with different incubation durations.

关 键 词：脂联素 脂肪酸合成酶(FAS) 激素敏感性脂肪酶(HSL) HEPG2细胞 启动子活性 

分 类 号：R589[医药卫生—内分泌]