利用重组腺病毒过表达<em>PPARγ</em>对牛肌肉细胞脂质沉积的影响

doi:10.3969/j.issn.1674-7968.2020.03.010

摘要
图/表
参考文献
相关文章 (15)

全文: PDF (6807 KB) HTML (1 KB)
输出: BibTeX | EndNote (RIS)

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 receptor γ, 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pAdTrack-CMV-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, qRT-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 (PPARγ coactivator-1 α, PGC1a)的表达水平上调至141.41倍(P≤0.01),而生肌因子5 (myogenic factor 5, Myf5)的表达变化不显著。本研究成功包装了PPARγ腺病毒,为进一步研究该基因的功能提供了工具,同时证明在牛肌肉细胞中过表达PPARγ有促进肌肉细胞内脂质沉积的趋势。

	服务

	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	滑留帅
	王璟
	徐照学
	王二耀
	陈宏

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

Abstract：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 PPARγ gene 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 293A 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 (qRT-PCR). 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 (Nf2f2) were down-regulated to 0.45, 0.57 and 0.25 times (P≤0.05). The expression level of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1a) was up-regulated to 141.41 times (P≤0.01), and the expression of myogenic factor 5 (Myf5) gene was not changed much. This study successfully packaged PPARγ Adenovirus, providing a tool for further study of the function of this gene, and proved that overexpression of PPARγ in bovine muscle cells has a tendency to promote fat deposition in muscle cells.

Key words： Adenovirus Peroxisome proliferator-activated receptor γ (PPARγ) gene Over-expression Muscle cell Fat deposition qRT-PCR

收稿日期: 2019-07-05

ZTFLH:	S823
	S-3

基金资助: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)

通讯作者: *chenhong1212@263.net;wangeryao666@qq.com

引用本文:

滑留帅, 王璟, 徐照学, 王二耀, 陈宏. 利用重组腺病毒过表达PPARγ对牛肌肉细胞脂质沉积的影响[J]. 农业生物技术学报, 2020, 28(3): 475-482.
HUA Liu-Shuai, WANG Jing, XU Zhao-Xue, WANG Er-Yao, CHEN Hong. Effects of PPARγ Overexpression by Recombinant Adenovirus on Fat Deposition in Cattle (Bos taurus) Muscle Cells. 农业生物技术学报, 2020, 28(3): 475-482.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2020.03.010 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2020/V28/I3/475

1 陈健华, 孙颖, 段友容. 2017. 静脉递送腺病毒载体在肿瘤基因治疗中的研究进展[J]. 肿瘤, 37(12): 1339-1343.
(Chen J H, Sun Y, Duan Y R.2017. Advances in intravenous delivery of adenoviral vectors in tumor gene therapy[J]. Tumor, 37(12): 1339-1343.)
2 崔婷婷, 邢天宇, 褚衍凯, 等. 2017. PPARγ在脂肪生成中的遗传和表观遗传调控[J]. 遗传, 39(11): 1066-1077.
(Cui T T, Xing T Y, Chu Y K, et al.2017. Genetic and epigenetic regulation of PPARγ during adipogenesis[J]. Hereditas, 39(11): 1066-1077.)
3 段安琴, 庞春英, 朱鹏, 等. 2016. 腺病毒载体转染水牛不同原代体细胞的效果比较[J]. 中国畜牧兽医, 43(10): 2661-2665.
(Duan A Q, Pang C Y, Zhu P, et al.2016. Effect comparison of Adenovirus vector transfer into different buffalo cells[J]. China Animal Husbandruy & Veterinary Medicine, 43(10): 2661-2665.)
4 郝称莉, 李齐发, 乔永, 等. 2008. 湖羊肌肉组织H-FABP和PPARγ基因表达水平与肌内脂肪含量的相关研究[J]. 中国农业科学, 4(11): 3776-3783.
(Hao C L, Li Q F, Qiao Y, et al.2008. Association of the H-FABP and PPARγ gene expression with intramuscular fat content in Hu sheep muscles[J]. Scientia Agricultura Sinica, 4(11): 3776-3783)
5 滑留帅. 2012. 牛SHH基因通过PPARγ通路调控脂肪生成[D]. 博士学位论文, 西北农林科技大学, 导师: 陈宏, pp. 43-47.
(Hua L S.2012. Cattle SHH gene regulating adipogenesis through PPARγ pathway[D]. Thesis for Ph.D., Northwest A&F University, Supervisor: Chen H, pp. 43-47.)
6 滑留帅, 王璟, 李明勋, 等. 2013. 动物脂肪组织的分化起源[J]. 中国牛业科学, 39(1): 42-45.
(Hua L S, Wang J, Li M X, et al.2013. The differentiation orgin of animal adipose[J]. China Cattle Science, 39(1): 42-45.)
7 黄锦亮. 2012. 猪PPARγ2基因肌肉超表达真核表达载体构建以及在转基因小鼠中的功能验证[D]. 博士学位论文, 华中农业大学, 导师: 熊远著, pp. 64-68.
(Huang J L.2012. Construction of swine muscle-specific overexpression PPARγ2 eukaryotic-expressing vectors and the function verification in transgenic mice[D]. Thesis for Ph.D., Huazhong Agricultural University, Supervisor: Xiong Y Z, pp. 64-68.)
8 李健. 2010. 西杂牛PPARγ2、PGC-1α、MEF2C基因表达量及其与肌内脂肪含量、嫩度的相关分析[D]. 硕士学位论文, 四川农业大学, 导师: 赖松家, pp. 25-36.
(Li J.2010. The gene expression of PPARγ2, PGC-1α, MEF2C and their correlation analysis with intramuscular fat content and tenderness in Simmental hybridization[D]. Thesis for M.S., Sichuan Agricultural University, Supervisor: Lai S J, pp. 25-36)
9 李琳, 郑卓, 贺红专. 2016. PPARγ基因调控猪脂肪沉积研究进展[J]. 中国猪业, 11(08): 57-60.
(Li L, Zheng Z, He H Z.2016. Progress in the regulation of porcine fat deposition by PPARγ gene[J]. China Swine Industry, 11(08): 57-60.)
10 李密杰, 张春梅, 蓝贤勇, 等. 2012. 共激活因子PGC-1α的研究进展[J]. 中国牛业科学, 38(04): 35-38.
(Li M J, Zhang C M, Lan X Y, et al.2012. Research advance of coregulator PGC-1α[J]. China Cattle Science, 38(4): 35-38.)
11 李树国, 牛化欣, 于建华, 等. 2018. 肉牛肌内脂肪和脂肪酸的营养价值及其调控[J]. 黑龙江畜牧兽医, (21): 59-62.
(Li S G, Niu H X, Yu J H, et al. 2018. Nutritional value and regulation of intramuscular fat and fatty acid in beef cattle[J]. Heilongjiang Animal Science and Veterinary Medicine, (21): 59-62.)
12 赖新生. 2013. 牛DEC1基因抑制肌卫星细胞和脂肪细胞分化[D]. 博士学位论文, 西北农林科技大学, 导师: 陈宏, pp. 21-23.
(Lai X S.2013. Cattle DEC1 gene inhibits defferentiation of satellite cells and preadipocyte[D]. Thesis for Ph.D., Northwest A&F University, Supervisor: Chen H, pp. 21-23.)
13 李优磊. 2018. PPAR信号通路在调控猪皮下脂肪与肌内脂肪差异沉积中的作用及机制研究[D]. 博士学位论文, 西北农林科技大学, 导师: 杨公社; 郑雪莉; 李晓, pp. 45-52.
(Li Y L.2018. The differential function and mechanism of PPAR signaling in regulation of porcine subcutaneous and intramuscular fat deposition[D]. Thesis for Ph.D., Northwest A&F University, Supervisor: Yang G S, Zheng X L, Li X, pp. 45-52.)
14 毛衍伟, 张一敏, 朱立贤, 等. 2018. 肌内脂肪对牛背最长肌品质的影响及肌内脂肪沉积机制的研究[J]. 食品与发酵工业, 44(11): 89-96.
(Mao Y W, Zhang Y M, Zhu L X, et al.2018. Effect of intramuscular fat on longissimus dorsi muscle quality and mechanism of intramuscular fat deposition study[J]. Food and Fermentation Industries, 44(11): 89-96.)
15 应飞. 2016. 利用转基因猪模型研究PGC1α对肌纤维类型转变的影响及其分子机制[D]. 博士学位论文, 华中农业大学, 导师: 熊远著, 左波, pp. 12-17 (Ying F. 2016. The effects of PGC1α gene on the muscle fiber conversion and their molecular mechanism in transgenic pig model[D]. Thesis for Ph.D., Huazhong Agriculural University, Supervisor: Xiong Y Z, Zuo B, pp. 12-17.)
16 张乐, 宁越, 李佩韦, 等. 2018. 腺病毒载体介导Smad3基因在秦川牛前体脂肪细胞中的过表达和沉默[J]. 畜牧兽医学报, 49(9): 1840-1850.
(Zhang L, Ning Y, Li P W, et al.2018. The overexpression and interference of Smad3 gene in Qinchuan cattle preadipocytes mediated by Adenovirus vector[J]. Chinese Journal of Animal and Veterinary Sciences, 49(9): 1840-1850.)
17 Asakura A, Rudnicki M A, Komaki M.2001. Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation[J]. Differentiation, 68(4): 245-253.
18 Esterbauer H, Oberkofler H, Krempler F, et al.1999. Human peroxisome proliferator activated receptor gamma coactivator 1 (PPARGC1) gene: cDNA sequence, genomic organization, chromosomal localization, and tissue expression[J]. Genomics, 62(1): 98-102.
19 Hausman G J, Dodson M V, Ajuwon K, et al.2009. The biology and regulation of preadipocytes and adipocytes in meat animals[J]. Journal of Animal Science, 87(4): 1218-1246.
20 Luo J, Deng Z, Luo X, et al.2007. A protocol for rapid generation of recombinant Adenoviruses using the AdEasy system[J]. Nature Protocols, 2(5): 1236-1247.
21 Ramakrishnan M A.2016. Determination of 50% endpoint titer using a simple formula[J]. World Journal of Virology, 5(2): 85-86.
22 Zeng Z, Yu B, Mao X, et al.2012. Effects of dietary digestible energy concentration on growth, meat quality, and PPARγamma gene expression in muscle and adipose tissues of Rongchang piglets[J]. Meat science, 90(1): 66-70.