利用重组腺病毒过表达PPARγ对牛肌肉细胞脂质沉积的影响  

Effects of PPARγ Overexpression by Recombinant Adenovirus on Fat Deposition in Cattle(Bos taurus) Muscle Cells

作　　者：滑留帅[1,2] 王璟[2] 徐照学[2] 王二耀[2] 陈宏[1] HUA Liu-Shuai;WANG Jing;XU Zhao-Xue;WANG Er-Yao;CHEN Hong(College of Animal Science and Technology,Northwest A&F University/Shaanxi Key Laboratory of Molecular Biology for Agriculture,Yangling 712100 China;Henan Key Laboratory of Farm Animal Breeding and Nutritional Regulation/Institute of Animal Husbandry and Veterinary Science,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China)

机构地区：[1]西北农林科技大学动物科技学院/陕西省农业分子生物学重点实验室,杨凌712100 [2]河南省农业科学院畜牧兽医研究所/河南省畜禽繁育与营养调控重点实验室,郑州450002

出　　处：《农业生物技术学报》2020年第3期475-482,共8页Journal of Agricultural Biotechnology

基　　金：国家肉牛牦牛产业技术体系(CARS-37);河南省肉牛产业技术体系(S2013-08)。

摘　　要：动物肌内脂肪沉积是一个复杂的生物学过程,涉及肌肉细胞分化、脂肪细胞分化及甘油三酯代谢等多个方面。过氧化物酶体增殖物活化受体γ(peroxisome proliferator-activated receptorγ, PPARγ)基因在动物的脂肪细胞分化过程中具有核心调控作用,但是PPARγ基因的表达水平与肌内脂肪含量之间的关系尚不明确。本研究旨在包装牛(Bos taurus) PPARγ基因腺病毒(Adenovirus),并分析过表达PPARγ对肌肉细胞脂质沉积的影响。将PPARγ亚克隆至腺病毒穿梭载体pAdTrack-CMV后,构建载体pAdTrackCMV-PPARγ。将线性化的pAdTrack-CMV-PPARγ载体和腺病毒骨架载体pAdEasy-1共转化至重组细菌大肠杆菌(Escherichia coli) BJ5183细胞中,在细菌中同源重组后,成功构建腺病毒骨架载体pAdEasy-1-PPARγ。将线性化的pAdEasy-1-PPARγ转入人(Homo sapiens) 293A细胞中包装并扩增PPARγ腺病毒。用PPARγ腺病毒感染牛肌肉细胞后,用实时荧光定量PCR (quantitative real-time PCR, q RT-PCR)检测脂质沉积相关基因的表达变化。结果表明,成功构建了PPARγ腺病毒载体,包装了PPARγ腺病毒并进行2次扩增后,获得的PPARγ腺病毒滴度为2×1011空斑形成单位(plaque forming unit, PFU)/mL。使用该PPARγ腺病毒感染牛肌肉细胞,获得了90%以上的感染效率。在牛肌肉细胞中过表达PPARγ后,脂质沉积正向调控基因脂肪酸结合蛋白4 (fat acid binding proteins 4, FABP4)、葡萄糖转运蛋白4 (glucose transporter 4, Glut4)和脂肪特异性磷脂酶A2 (adipose-specific phospholipase A2, AdPLA2)的表达水平分别上升至1.89、1.51和1.56倍(P≤0.05)。而脂质沉积负调控基因GATA结合蛋白2 (GATA binding protein 2,GATA2)、激素敏感脂酶(hormone-sensitive lipase, HSL)、核受体亚家族2F组成员2 (nuclear receptor subfamily 2, group F, member 2, Nr2f2)的表达水平分别下降至0.45、0.57和0.25倍(P≤0.05)。同时,PPARγ辅激活因子1α(PPARγcoactivator-1α, PGC1a)的表达水平�Intramuscular fat deposition is a complex biological process involving muscle cell differentiation,adipocyte differentiation and triglyceride metabolism. Peroxisome proliferator-activated receptor γ(PPARγ)gene plays a central role in the differentiation of adipocytes in animals, but the relationship between PPARγgene expression and intramuscular fat content still not clear. The aim of this study was to package the cattle(Bos taurus) PPARγ Adenovirus and analyze the effects of PPARγ overexpression on fat deposition in muscle cells. After subcloning PPARγ into the vector pAdTrack-CMV, a shuttle vector pAdTrack-CMV-PPARγ was constructed. The linearized shuttle vector pAdTrack-CMV-PPARγ and the adenoviral backbone vector pAdEasy-1 were co-transformed into the recombinant bacterial BJ5183(Escherichia coli), and after homologous recombination, the adenoviral backbone vector pAdEasy-1-PPARγ containing the gene of interest was constructed. The linearized pAdEasy-1-PPARγ was transferred into 293 A cells(Homo sapiens) to package and amplify the PPARγ Adenovirus. After infection of bovine muscle cells with PPARγ Adenovirus, the expression changes of the fat deposition related genes were detected by quantitative real-time PCR(qRTPCR). The results showed that the PPARγ Adenovirus vector was successfully constructed. After the package and 2 times of amplification, the PPARγ Adenovirus with a titer of 2×1011 plaque forming unit(PFU)/mL were obtained. Infection of bovine muscle cells with the PPARγ Adenovirus yielded an infection efficiency of more than 90%. After overexpression of PPARγ in bovine muscle cells, the positive regulation genes of fat deposition, including fat acid binding proteins 4(FABP4), glucose transporter 4(Glut4) and adipose-specific phospholipase A2(AdPLA2) were up-regulated to 1.89, 1.51 and 1.56 times(P≤0.05). While the negative regulation genes of fat deposition including GATA binding protein 2(GATA2), hormone-sensitive lipase(HSL)and nuclear receptor subfamily 2, group F, member 2(Nf2

关 键 词：腺病毒 过氧化物酶体增殖物活化受体γ基因(PPARγ) 过表达 肌肉细胞 脂质沉积 qRT-PCR 

分 类 号：S823[农业科学—畜牧学] S-3[农业科学—畜牧兽医]