KLF9基因腺病毒载体的构建及其脂肪酸氧化功能

KLF9 adenoviral vector construction and it's function in fatty acid oxidation

目的:构建Krüppel样转录因子9(KLF9)过表达的腺病毒,初步探讨KLF9在肝脏脂肪酸氧化中的功能。方法:根据分子克隆的原理,构建融合表达3×Flag的KLF9过表达的腺病毒,并在293A细胞中验证KLF9腺病毒感染效率。将融合表达3×Flag的KLF9过表达的腺病毒分别感染肝原代细胞、正常饮食小鼠以及高脂小鼠,通过Q-PCR检测过氧化物酶体增殖物活化受体-γ协同刺激因子PGC 1-α等脂肪酸氧化相关基因的表达情况。结果:Q-PCR以及Western blot结果证明融合表达3×Flag的KLF9过表达腺病毒载体包装成功,通过荧光显微镜观察到感染效率达90%以上。经Q-PCR检测,感染Ad-KLF9的肝原代细胞和肝脏组织中,PGC1-α等脂肪酸氧化相关基因表达明显高于对照组。结论:初步认定KLF9可促进脂肪酸氧化,改善高脂诱导的非酒精性脂肪肝。

Objective:To construct Krüppel-like transcription factor 9(KLF9)overexpressed adenovirus and explore the role of KLF9 in liver fatty acid oxidation.Methods:KLF9 fusion with 3×Flag overexpressed adenovirus was constructed,according to the principle of molecular cloning,and the infection efficiency of adenovirus was tested in 293A cells.The Ad-KLF9 was infected into primary liver cells,chow and high-fat diet induced mice respectively,and the expression of fatty acid oxidation related genes such as PGC1-αwere detected by Q-PCR.Results:Q-PCR and Western blot results showed that KLF9 fusion with 3×Flag overexpressed adenovirus was successfully packaged,and more than 90%of the infection efficiency was observed by fluorescence microscopy.The expression of fatty acid oxidation related genes such as PGC1-αin primary liver cells and liver tissues infected with Ad-KLF9 were significantly higher than those in the control group by Q-PCR.Conclusion:It is preliminarily determined that KLF9 can promote fatty acid oxidation and improve the non-alcoholic fatty liver disease.