转录因子ChREBP-α增强小鼠原代肝细胞的脂质合成能力  

Transcription factor ChREBP-α enhances lipogenesis of primary hepatocytes

作　　者：周露婷[1] 杨瑞[1] 李玲[1] 陈榕[1,2] 张晔[1] 印慨[3] 邹大进[2] 张海[1] 章卫平[1] 

机构地区：[1]第二军医大学基础部病理生理学教研室,上海200433 [2]第二军医大学长海医院内分泌科,上海200433 [3]第二军医大学长海医院普通外科,上海200433

出　　处：《第二军医大学学报》2013年第1期6-10,共5页Academic Journal of Second Military Medical University

基　　金：国家杰出青年科学基金(31025013);国家自然科学基金(81100614)~~

摘　　要：目的构建含转录因子ChREBP-α基因的重组腺病毒载体,检测其在小鼠原代肝细胞中的表达及其对脂质合成的调节作用。方法将ChREBP-αcDNA克隆到穿梭质粒pShuttle-CMV载体,与pAdEasy质粒在大肠杆菌BJ5183中同源重组,获得腺病毒载体pAd-ChREBP-α,在293A细胞中进行病毒的包装和扩增,检测病毒的滴度;将腺病毒Ad-ChREBP-α感染小鼠原代肝细胞,用实时荧光定量PCR及蛋白质印迹法检测ChREBP-α的表达,实时荧光定量PCR检测ChREBP-α靶基因LPK mRNA的表达。用核素14 C示踪法测定肝细胞的脂质合成速率。结果成功制备了ChREBP-α重组腺病毒,利用其在原代肝细胞过表达ChREBP-α,可显著上调靶基因LPK的表达,并增强肝细胞的脂质合成速率。结论成功制备了具有生物学活性、能在原代肝细胞中过表达的ChREBP-α重组腺病毒,为研究ChREBP-α的糖脂代谢调控作用奠定了基础。Objective To construct recombinant adenovirus expressing mouse ChREBP-a, and examine the effect of ChREBP-a overexpression on lipid synthesis in mouse primary hepatoeytes. Methods The mouse ChREBP-a cDNA was subcloned into pShuttle-CMV vector, and the product was transformed into E. colt strain BJ5183 for homologous recombination with pAdEasy-1. The resultant recombinant vector was transfected into 293A cells for viral package. Mouse primary hepatocytes were infected with adenoviruses Ad-ChREBP-a, and gene expression was analyzed at mRNA and protein levels by real-time PCR and Western blotting analsysis. The expression levels of ChREBP target gene LPK were measured at mRNA level by real- time PCR, and the fatty acid synthesis rate was determined by [^14C]-acetate incorporation. Results We successfully constructed recombinant adenoviruses expressing mouse ChREBP-a. Adenovirus-mediated overexpression of ChREBP-a markedly increased LPK mRNA expression and fatty acid synthesis in primary hepatocytes. Conclusion We have successfully constructed recombinant adenoviruses expressing mouse ChREBP-a, which is biologically active and can overexpress ChREBP-a in primary hepatocytes. Overexpression of ChREBP-a can enhance lipid synthesis.

关 键 词：脂代谢障碍 重组腺病毒 肝细胞 碳水化合物反应元件结合蛋白 

分 类 号：R589.2[医药卫生—内分泌]