36型人腺病毒E4ORF1真核表达载体的构建及其促进前脂肪细胞3T3-L1对葡萄糖利用的作用初探  

Construction of eukaryotic expression vector of human adenovirus type 36 E4ORF1 and its effect on promoting glucoseutilization by 3T3-L1 preadipocytes

作　　者：王冰丽 陈哲 艾柯代姆·阿卜杜克热木 刘迪晖 赵炳尧 关亚群 WANG Bingli;CHEN Zhe;Aikedaimu Abudukeremu;LIU Dihui;ZHAO Bingyao;GUAN Yaqun(Department of Biochemistry and Molecular Biology,School of Basic Medicine,Xinjiang Medical University,The State Key Laboratory of Pathogenesis,Prevention and Treatment of High Incidence Diseases in Central Asia,Urumqi 830011,China)

机构地区：[1]新疆医科大学基础医学院生物化学与分子生物学教研室,省部共建中亚高发病成因与防治国家重点实验室,乌鲁木齐830011

出　　处：《新疆医科大学学报》2022年第5期469-478,共10页Journal of Xinjiang Medical University

基　　金：国家自然科学基金面上项目(81873664)。

摘　　要：目的通过构建早期基因4开放读码框1(E4ORF1)真核表达载体,探讨E4ORF1蛋白调节前脂肪细胞3T3-L1(小鼠胚胎成纤维细胞)利用葡萄糖的作用。方法通过分子克隆技术构建pcDNA3.1-E4ORF1重组质粒,并进行DNA测序鉴定。将前脂肪细胞3T3-L1分为对照组和E4ORF1转染组,检测两组细胞第0、1、2、3天培养基中葡萄糖浓度,利用RT-qPCR和Western-Blot检测两组细胞第0、1、2、3、4天E4ORF1、己糖激酶2(HK2)、糖原合酶1(GYS1)mRNA和蛋白质的表达情况。油红O染色观察两组细胞第0、1、2、3、4天分化及脂质积累情况,检测过氧化物酶体增殖物激活受体γ(PPARγ)、CCAAT增强子结合蛋白a(C/EBPa)、脂肪酸结合蛋白4(FABP4)、脂肪酸合成酶(FASN)、乙酰辅酶A羧化酶(ACC)mRNA和蛋白质的表达情况。结果成功构建N端带FLAG(DYKDDDDK肽)标签的真核表达载体pcDNA3.1-E4ORF1。与对照组相比,E4ORF1转染组细胞培养基中葡萄糖浓度随培养时间延长下降更快,差异有统计学意义(P<0.01)。RT-qPCR和Western-Blot结果显示,与对照组相比,转染组E4ORF1的mRNA和蛋白表达水平均显著增高,差异有统计学意义(P<0.05)。E4ORF1转染组的糖酵解关键酶基因HK2和糖原合酶基因GYS1 mRNA和蛋白质的表达均显著上升(P均<0.05)。油红O染色显示两组细胞均无成脂分化现象,脂肪细胞分化标志基因PPARγ、C/EBPa、FABP4、FASN、ACC的mRNA和蛋白表达水平均无显著变化(P>0.05)。结论成功构建pcDNA3.1-E4ORF1真核表达载体;在前脂肪细胞3T3-L1中,E4ORF1可能通过促进HK2和GYS1的表达,参与细胞对葡萄糖的分解和糖原合成过程,对前脂肪细胞的分化无影响。Objective To explore the role of early gene 4 open reading frame 1(E4ORF1) protein in regulating glucose utilization of preadipocytes 3T3-L1(Mouse embryonic fibroblasts) by constructing E4ORF1 eukaryotic expression vector. Methods The full length of E4ORF1 coding region was obtained by molecular cloning technology, and it was ligated into pcDNA3.1(-) eukaryotic expression vector, and recombinant plasmid was identified by DNA sequencing. The preadipocytes 3T3-L1 were divided into the control group and the E4ORF1 transfection group, and the glucose concentration in the culture medium of the two groups of cells on 0, 1, 2, and 3 days, and the cells detected by RT-qPCR and Western-Blot for expression of E4ORF1, Hexokinase 2(HK2) and Glycogen synthase 1(GYS1) mRNA and protein on day 0, 1, 2, 3, and 4 of the two groups of cells. Oil red O staining was used to observe the differentiation and the lipid accumulation of the cells in the two groups on days 0, 1, 2, 3, and 4. Peroxisome proliferator-activated receptor(PPARγ), CCAAT enhancer-binding protein(C/EBPa), fatty acid binding protein 4(FABP4), fatty acid synthase(FASN), acetyl-CoA carboxylase(ACC) mRNA and protein expression. Results The eukaryotic expression vector pcDNA3.1-E4ORF1 with N-terminal FLAG(DYKDDDDK peptide) tag was successfully constructed. Compared with the control group, the glucose concentration in the cell culture medium of E4ORF1 transfection group was decreased faster with prolongation in culture time, and the difference was statistically significant(P<0.01). RT-qPCR and WesternBlot results showed that compared with the control group, mRNA and protein expression levels of E4ORF1 in the transfection group were significantly increased, and the difference was statistically significant(P<0.05). The mRNA and protein expressions of glycolysis key enzyme gene HK2 and glycogen synthase gene GYS1 in the E4ORF1 transfection group were significantly increased(all P<0.05). Oil red O staining showed that there was no adipogenic differentiation in the two group

关 键 词：36型人腺病毒 早期基因4开放读码框1 己糖激酶 前脂肪细胞3T3-L1 糖代谢 

分 类 号：R551.8[医药卫生—血液循环系统疾病] Q591.4[医药卫生—内科学]