山羊APOA 4基因抑制肌内脂肪细胞分化  

APOA 4 Gene Inhibits Intramuscular Adipocyte Differentiation in Goat

作　　者：曲比伍且 李艳艳 李鑫[1,2] 王永[1,2] 王友利 刘伟[1,2] 朱江江[1,2] 林亚秋[1,2] QUBI Wuqie;LI Yanyan;LI Xin;WANG Yong;WANG Youli;LIU Wei;ZHU Jiangjiang;LIN Yaqiu(Key Laboratory of Protection and Utilization of Animal Genetic Resources on Qinghai-Tibet Plateau of Ministry of Education/Sichuan Province,Southwest Minzu University,Chengdu 610041,China;College of Animal Science and Veterinary Medicine,Southwest Minzu University,Chengdu 610041,China)

机构地区：[1]西南民族大学青藏高原动物遗传资源保护与利用教育部/四川省重点实验室,成都610041 [2]西南民族大学畜牧兽医学院,成都610041

出　　处：《畜牧兽医学报》2023年第5期1927-1938,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(32072723,31902154);四川省应用基础研究计划重点项目(2022JDTD0030);西南民族大学研究生创新型科研项目(ZD2022683)。

摘　　要：旨在明确山羊脂蛋白A-IV(apolipoprotein A-IV,APOA 4)对肌内脂肪细胞分化的调控作用。本试验所用动物为1周岁生理状态良好,舍饲的简州大耳羊公羊(n=5)。试验利用RT-PCR技术克隆山羊APOA 4基因序列,通过生物信息学分析方法对克隆得到的序列进行分析,并获得序列特征;利用qPCR技术构建山羊APOA 4的组织与细胞时序表达谱;通过构建山羊APOA 4基因过表达载体,本试验分为过表达组(pEGFP-N1-APOA 4)与阴性对照组(pEGFP-N1),每组设置3个重复,每个重复的样本量为2个。转染pEGFP-N1-APOA 4过表达载体和pEGFP-N1至山羊肌内前体脂肪细胞并诱导分化,通过油红O染色、Bodipy染色、DAPI染色、吸光度(OD490 nm)测定及qPCR等方法确定过表达山羊APOA 4基因对肌内脂肪细胞分化及脂肪形成标志物的影响。结果表明:1)试验克隆得到山羊APOA 4基因序列1467 bp,其中CDS区1143 bp,编码380个AA;2)生物信息学分析推测得到山羊APOA4为带负电且属于酸性不稳定疏水蛋白;3)山羊APOA 4在山羊各组织广泛表达,在肝中表达量最高,极显著高于其他组织(P<0.01)。APOA 4在诱导分化48 h的肌内脂肪细胞中表达最高;4)过表达山羊APOA 4基因后与对照组相比较,发现肌内脂肪细胞脂滴积聚减少,脂肪分化标志基因C/EBPα、PPARγ表达极显著下调(P<0.01),SREBP1、C/EBPβ表达水平显著下调(P<0.05)。脂代谢标志基因AP2、LPL显著下调(P<0.05)。综合形态学染色和分子生物学结果,山羊APOA 4基因可能通过抑制分化标志基因C/EBPα、PPARγ、SREBP1、C/EBPβ以及脂代谢标志基因AP2、LPL的表达,进而抑制肌内脂肪细胞的分化和脂滴积聚,提示过表达山羊APOA 4基因抑制肌内脂肪细胞分化。The study aimed to determine the regulatory effect of goat apolipoprotein A-IV(APOA 4)on intramuscular adipocyte differentiation.The animals used in this experiment were 1 year old Jianzhou big ear goat rams(n=5)with good physiological state.The APOA 4 gene sequence of goat was cloned by RT-PCR,and the cloned sequence was analyzed by bioinformatics analysis,and the sequence characteristics were obtained.Temporal expression profiles of APOA 4 in tissues and cells of goat were constructed by qPCR.By constructing the goat APOA 4 gene overexpression vector,this experiment was divided into an overexpression group(pEGFP-N1-APOA 4)and a negative control group(pEGFP-N1),with 3 replicates in each group and 2 samples in each replicate.Transfected pEGFP-N1-APOA 4 overexpression vector and pEGFP-N1 into goat intramuscular precursor adipocytes and induced differentiation.Oil red O staining,Bodipy staining,DAPI staining,absorbance(OD490 nm)and qPCR were used to determine the effects of overexpression of goat APOA 4 gene on adipocyte differentiation and adipogenesis markers.The results showed as follows:1)The APOA 4 gene sequence of goat was 1467 bp,in which the CDS region was 1143 bp,encoding 380 AA.2)Bioinformatics analysis predicted that APOA4 was negatively charged and acid-unstable hydrophobic protein.3)APOA 4 was widely expressed in goat tissues,and the highest expression level was found in liver,which was significantly higher than that in other tissues(P<0.01).APOA 4 expression was the highest in intramuscular adipocytes at 48 h after induction of differentiation.4)Compared with the control group after APOA 4 gene overexpression,the accumulation of lipid droplets in intramuscular adipocytes was decreased,and the expression levels of adipose-differentiation markers C/EBPαand PPARγwere significantly down-regulated(P<0.01),and the expression levels of SREBP 1 and C/EBPβwere significantly down-regulated(P<0.05).Lipid metabolism marker genes AP 2 and LPL were significantly down-regulated(P<0.05).Based on morphological stai

关 键 词：山羊 APOA 4 肌内脂肪细胞 过表达 细胞分化 

分 类 号：S827.2[农业科学—畜牧学]