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| 转录因子ChREBP-α增强小鼠原代肝细胞的脂质合成能力 |
| 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* |
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(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
△共同第一作者
*通信作者) |
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| 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-α cDNA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14C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。 |
| 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 |
| DOI：10.3724/SP.J.1008.2013.006 |
| 投稿时间：2012-11-12修订日期：2012-12-26 |
| 基金项目:国家杰出青年科学基金(31025013),国家自然科学基金(81100614). |
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| Transcription factor ChREBP-α enhances lipogenesis of primary hepatocytes |
| ZHOU Lu-ting1△,YANG Rui1△,LI Ling1,CHEN Rong1,2,ZHANG Ye1,YIN Kai3,ZOU Da-jin2,ZHANG Hai1,ZHANG Wei-ping1* |
(1. Department of Pathophysiology, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, China
2. Department of Endocrinology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
3. Department of General Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
△Co-first authors.
*Corresponding author.) |
| Abstract: |
| Objective To construct recombinant adenovirus expressing mouse ChREBP-α, and examine the effect of ChREBP-α overexpression on lipid synthesis in mouse primary hepatocytes. Methods The mouse ChREBP-α cDNA was subcloned into pShuttle-CMV vector, and the product was transformed into E. coli 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-α, 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-α. Adenovirus-mediated overexpression of ChREBP-α markedly increased LPK mRNA expression and fatty acid synthesis in primary hepatocytes. Conclusion We have successfully constructed recombinant adenoviruses expressing mouse ChREBP-α, which is biologically active and can overexpress ChREBP-α in primary hepatocytes. Overexpression of ChREBP-α can enhance lipid synthesis. |
| Key words: lipid metabolism disorders recombinant adenovirus hepatocytes carbohydrate response element binding protein |
