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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 a recombinant adenovirus vector containing the transcription factor ChREBP-α gene and test its expression in mouse primary hepatocytes and its regulation on lipid synthesis. Methods ChREBP-α cDNA was cloned into the shuttle plasmid pShuttle-CMV vector and homologously recombined with pAdEasy plasmid in E. coli BJ5183 to obtain the adenovirus vector pAd-ChREBP-α. The virus was packaged and amplified in 293A cells and the virus was detected The primary hepatocytes of mice were infected with adenovirus Ad-ChREBP-α, the expression of ChREBP-α was detected by real-time fluorescence quantitative PCR and Western blotting, and the expression of LPK mRNA of ChREBP-α target gene was detected by real-time fluorescence quantitative PCR . The rate of lipid synthesis in hepatocytes was determined by radionuclide 14 C tracer method. Results ChREBP-α recombinant adenovirus was successfully prepared. ChREBP-α was overexpressed in primary hepatocytes, which significantly up-regulated the expression of LPK and enhanced the rate of lipid synthesis in hepatocytes. Conclusion ChREBP-α recombinant adenovirus, which has biological activity and can be overexpressed in primary hepatocytes, was successfully prepared and laid the foundation for the study on the regulation of glycolipid metabolism by ChREBP-α.