山羊APOA4基因抑制肌内脂肪细胞分化

doi:10.11843/j.issn.0366-6964.2023.05.015

摘要/Abstract

摘要： 旨在明确山羊脂蛋白A-IV(apolipoprotein A-IV,APOA4)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APOA4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APOA4的组织与细胞时序表达谱;通过构建山羊APOA4基因过表达载体,本试验分为过表达组(pEGFP-N1-APOA4)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- APOA4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APOA4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APOA4基因序列1 467 bp,其中CDS区1 143 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APOA4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)。APOA4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APOA4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APOA4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APOA4基因抑制肌内脂肪细胞分化。

关键词: 山羊, APOA4, 肌内脂肪细胞, 过表达, 细胞分化

Abstract: The study aimed to determine the regulatory effect of goat apolipoprotein A-IV (APOA4) 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 APOA4 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 APOA4 in tissues and cells of goat were constructed by qPCR. By constructing the goat APOA4 gene overexpression vector, this experiment was divided into an overexpression group (pEGFP-N1- APOA4) and a negative control group (pEGFP-N1), with 3 replicates in each group and 2 samples in each replicate. Transfected pEGFP-N1-APOA4 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 APOA4 gene on adipocyte differentiation and adipogenesis markers. The results showed as follows:1) The APOA4 gene sequence of goat was 1 467 bp, in which the CDS region was 1 143 bp, encoding 380 AA. 2) Bioinformatics analysis predicted that APOA4 was negatively charged and acid-unstable hydrophobic protein. 3) APOA4 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). APOA4 expression was the highest in intramuscular adipocytes at 48 h after induction of differentiation. 4) Compared with the control group after APOA4 gene overexpression, the accumulation of lipid droplets in intramuscular adipocytes was decreased, and the expression levels of adipose-differentiation markers C/EBPα and PPARγ were significantly down-regulated (P< 0.01), and the expression levels of SREBP1 and C/EBPβ were significantly down-regulated (P<0.05). Lipid metabolism marker genes AP2 and LPL were significantly down-regulated (P<0.05). Based on morphological staining and molecular biology results, APOA4 gene may inhibit the differentiation of adipocytes and lipid droplet accumulation in goats by inhibiting the expression of differentiation markers C/EBPα, PPARγ, SREBP1, C/EBPβ and lipid metabolism markers AP2 and LPL. It was suggested that overexpression of APOA4 gene inhibited the differentiation of intramuscular adipocytes.

Key words: goat, APOA4, intramuscular adipocytes, overexpression, cell differentiation

中图分类号:

S827.2

曲比伍且, 李艳艳, 李鑫, 王永, 王友利, 刘伟, 朱江江, 林亚秋. 山羊APOA4基因抑制肌内脂肪细胞分化[J]. 畜牧兽医学报, 2023, 54(5): 1927-1938.

QUBI Wuqie, LI Yanyan, LI Xin, WANG Yong, WANG Youli, LIU Wei, ZHU Jiangjiang, LIN Yaqiu. APOA4 Gene Inhibits Intramuscular Adipocyte Differentiation in Goat[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 1927-1938.

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