实验方法 Experimental Methods

猪骨骼肌肌内脂滴分离和纯化方法的探究

展开
  • 1. 天津市农业科学院天津市畜牧兽医研究所, 天津 300381;
    2. 天津大学化工学院, 天津 300072;
    3. 西北农林科技大学动物科技学院, 杨凌 712100;
    4. 中国农业科学院北京畜牧兽医研究所, 动物营养学国家重点实验室, 北京 100193
闫峻(1984-),男,江苏徐州人,副研究员,博士,研究方向为脂代谢与肉品质调控。E-mail:yjjsxz@163.com

收稿日期: 2018-06-26

  网络出版日期: 2019-01-16

基金资助

国家自然科学基金青年科学基金项目(No.31402097);动物营养国家重点实验室开放基金(7008);天津市生猪产业体系创新团队资助(IT-TPRS2017005);中国博士后科学基金(No.2014M561191)

Isolation and Purification of Intramuscular Lipid Droplets in Porcine Skeletal Muscle

Expand
  • 1. Tianjin Institute of Animal Husbandry and Veterinary Medicine, Tianjin Academy of Agricultural Sciences, Tianjin 300381, China;
    2. Institute of Chemical Technology, Tianjin University, Tianjin 300072, China;
    3. College of Animal Science and Technology, Northwest A & F University, Yangling 712100, China;
    4. Institute of Animal Sciences of Chinese Academy of Agricultural Sciences State Key Laboratory of Animal Nutrition, Beijing 100193, China

Received date: 2018-06-26

  Online published: 2019-01-16

摘要

本试验旨在探究分离和纯化猪骨骼肌肌内脂滴的方法,为深入揭示肌内脂肪形成的调控机制奠定基础。试验采集体重为(120±2)kg的"杜×长×大"三元杂交猪的背最长肌,采用梯度超速离心法对猪背最长肌肌内脂滴初步超速分离,探索不同组织匀浆方式、缓冲液pH、温度及苯甲基磺酰氟(PMSF)对肌内脂滴初分离的影响,确定高效获取纯化脂滴的最佳离心力及离心时间。使用油红O及Bodipy染色进行肌内脂滴形态学鉴定,通过考马斯亮蓝染色及Western blot技术对脂滴进行纯度检测。结果表明:使用全自动组织研磨仪匀浆猪骨骼肌,在4℃、pH 7.4缓冲液中添加PMSF时分离获得的肌内脂滴形态完整;纯化条件为4℃、230 000×g离心30 min时获得脂滴量最多、形态最好,考马斯亮蓝染色鉴定脂滴蛋白条带丰富清晰,并且Western blot验证纯度最高。本实验室建立了猪骨骼肌肌内脂滴的分离和纯化方法,应用此方法获得了形态完整且纯度较高的肌内脂滴,该技术可为研究肌内脂肪生成和分子调控机制开辟新的途径。

本文引用格式

闫峻, 马衍旋, 穆淑琴, 唐湘方, 郑梓, 李宁, 刘陈龙, 李千军, 孙超 . 猪骨骼肌肌内脂滴分离和纯化方法的探究[J]. 动物营养学报, 2019 , 31(1) : 266 -273 . DOI: 10.3969/j.issn.1006-267x.2019.01.033

Abstract

This experiment aimed to explore the establishment of an optimal method for the isolation and purification of intramuscular lipid droplets from porcine skeletal muscle, which would lay the foundation for further revealing the regulation mechanism of intramuscular fat formation. Longissimus dorsi muscle tissue from body weight of (120±2) kg "Duroc×Landrace×Yorkshire" ternary hybrid finishing pigs was obtained after slaughter. The isolation of intramuscular lipid droplets of longissimus dorsi muscle was performed by ultracentrifugation. Explore the influence of different homogenization methods, temperature, buffer pH and phenylmethanesulfonyl fluoride (PMSF) on the isolation efficiency, and further determine the optimal centrifugal force and centrifugation time to purify lipid droplets. The results showed that the skeletal muscle was homogenized by a fully automatic tissue grinder, and the intramuscular lipid droplets were obtained by adding PMSF in 4℃, pH=7.4 buffer. With the purification conditions at 4℃, 230 000×g centrifugation with 30 min, the lipid droplets were the most abundant, the structure was complete, and the purity was the highest. In conclusion, the optimized isolation and purification method can obtain intramuscular lipid droplets of porcine skeletal muscles with complete morphology and high purity, which can open up new avenues to study intramuscular fat generation and the molecular regulation mechanism.

参考文献

[1] WOOD J D,RICHARDSON R I,NUTE G R,et al.Effects of fatty acids on meat quality:a review[J].Meat Science,2003,66(1):21-32.

[2] FERNANDEZ X,MONIN G,TALMANT A,et al.Influence of intramuscular fat content on the quality of pig meat-1.Composition of the lipid fraction and sensory characteristics of m. longissimus lumborum[J].Meat Science,1999,53(1):59-65.  

[3] 张恬,张龙超,王立刚,等.猪脂肪沉积候选基因AOC3、PPARG1及SOD3 DNA甲基化差异研究[J].中国畜牧兽医,2016,43(11):2820-2825.

[4] MURÄNI E,PONSUKSILI S,SEYFERT H M,et al.Dual effect of a single nucleotide polymorphism in the first intron of the porcine secreted phosphoprotein 1,gene:allele-specific binding of C/EBP beta and activation of aberrant splicing[J].BMC Molecular Biology,2009,10(1):96.

[5] ZHENG Q,LIN J,HUANG J J,et al.Reconstitution of UCP1 using CRISPR/Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity[J].Proceedings of the National Academy of Sciences of the United States of America,2017,114(45):E9474-E9482

[6] 张霞,刘晓研,苗义良.基因编辑技术在猪现代育种和动物模型构建中应用的研究进展[J].中国细胞生物学学报,2017,39(5):659-667.

[7] 张恬.猪肌内脂肪全基因组甲基化差异分析及候选基因研究[D].硕士学位论文.北京:中国农业科学院,2016:7-40.

[8] GANDOLFI G,MAZZONI M,ZAMBONELLI P,et al.Perilipin 1 and perilipin 2 protein localization and gene expression study in skeletal muscles of European cross-breed pigs with different intramuscular fat contents[J].Meat Science,2011,88(4):631-637.  

[9] MURPHY D J.The biogenesis and functions of lipid bodies in animals,plants and microorganisms[J].Progress in Lipid Research,2001,40(5):325-438.  

[10] FUJIMOTO T,OHSAKI Y,CHENG J L,et al.Lipid droplets:a classic organelle with new outfits[J].Histochemistry and Cell Biology,2008,130(2):263-279.  

[11] 张淑妍,杜雅兰,汪洋,等.脂滴-细胞脂类代谢的细胞器[J].生物物理学报,2010,26(2):97-105.

[12] WÄLTERMANN M,STEINBVCHEL A.Neutral lipid bodies in prokaryotes:recent insights into structure,formation,and relationship to eukaryotic lipid depots[J].Journal of Bacteriology,2005,187(11):3607-3619.  

[13] FEI W H,SHUI G H,GAETA B,et al.Fld1p,a functional homologue of human seipin,regulates the size of lipid droplets in yeast[J].Journal of Cell Biology,2008,180(3):473-482.  

[14] ZHANG P,NA H M,LIU Z Y,et al.Proteomic study and marker protein identification of Caenorhabditis elegans lipid droplets[J].Molecular & Cellular Proteomics,2012,11(8):317-328.  

[15] BELLER M,RIEDEL D,JANSCH L,et al.Characterization of the Drosophila lipid droplet subproteome[J].Molecular & Cellular Proteomics,2006,5(6):1082-1094.  

[16] TZEN J T C,PENG C C,CHENG D J,et al.A new method for seed oil body purification and examination of oil body integrity following germination[J].Journal of Biochemistry,1997,121(4):762-768.  

[17] 吴华莉,何大乾,王惠影,等.脂滴在动物组织中的功能[J].上海农业学报,2015(1):118-121.

[18] FARESE R V,Jr,WALTHER T C.Lipid droplets finally get a little R-E-S-P-E-C-T[J].Cell,2009,139(5):855-860.  

[19] SAMUEL V T,SHULMAN G I.Mechanisms for insulin resistance:common threads and missing links[J].Cell,2012,148(5):852-871.  

[20] DEBERARDINIS R J,THOMPSON C B.Cellular metabolism and disease:what do metabolic outliers teach us?[J].Cell,2012,148(6):1132-1144.  

[21] ZEHMER J K,HUANG Y G,PENG G,et al.A role for lipid droplets in inter-membrane lipid traffic[J].Proteomics,2009,9(4):914-921.  

[22] DING Y F,ZHANG S Y,YANG L,et al.Isolating lipid droplets from multiple species[J].Nature Protocols,2013,8(1):43-51.  

[23] MANNIK J,MEYERS A,DALHAIMER P.Isolation of cellular lipid droplets:two purification techniques starting from yeast cells and human placentas[J].Journal of Visualized Experiments Jove,2014(86),doi:10.3791/50981.

[24] OHSAKI Y,CHENG J L,SUZUKI M,et al.Biogenesis of cytoplasmic lipid droplets:from the lipid ester globule in the membrane to the visible structure[J].Biochimica et Biophysica Acta:Molecular and Cell Biology of Lipids,2009,1791(6):399-407.  

[25] YANG L,DING Y F,CHEN Y,et al.The proteomics of lipid droplets:structure,dynamics,and functions of the organelle conserved from bacteria to humans[J].Journal of Lipid Research,2012,53(7):1245-1253.  

[26] DING Y F,YANG L,ZHANG S Y,et al.Identification of the major functional proteins of prokaryotic lipid droplets[J].Journal of Lipid Research,2012,53(3):399-411.  

[27] JACQUIER N,CHOUDHARY V,MARI M,et al.Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae[J].Journal of Cell Science,2011,124(14):2424-2437.  

[28] PU J,HA C W,ZHANG S Y,et al.Interactomic study on interaction between lipid droplets and mitochondria[J].Protein & Cell,2011,2(6):487-496.  

[29] BRASAEMLE D L,WOLINS N E.Packaging of fat:an evolving model of lipid droplet assembly and expansion[J].Journal of Biological Chemistry,2012,287(4):2273-2279.  

[30] KRAHMER N,HILGER M,KORY N,et al.Protein correlation profiles identify lipid droplet proteins with high confidence[J].Molecular & Cellular Proteomics,2013,12(5):1115-1126.  

[31] STEIGEMANN P,GERLICH D W.Cytokinetic abscission:cellular dynamics at the midbody[J].Trends in Cell Biology,2009,19(11):606-616.  

[32] CHOI T,FUKASAWA K,ZHOU R,et al.The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes[J].Proceedings of the National Academy of Sciences of the United States of America,1996,93(14):7032-7035.  

[33] SEGER R,KREBS E G.The MAPK signaling cascade[J].The FASEB Journal,1995,9(9):726-35.  

[34] ZHANG H H,SOUZA S C,MULIRO K V,et al.Lipase-selective functional domains of perilipin A differentially regulate constitutive and protein kinase A-stimulated lipolysis[J].Journal of Biological Chemistry,2003,278(51):51535-51542.  

[35] GARCIA A,SUBRAMANIAN V,SEKOWSKI A,et al.The amino and carboxyl termini of perilipin a facilitate the storage of triacylglycerols[J].Journal of Biological Chemistry,2004,279(9):8409-8416.  

[36] 张峰.内质网分子伴侣Calnexin的研究进展[J].生物学通报,2008,43(8):7-10.

[37] PILCH P F,SOUTO R P,LIU L B,et al.Cellular spelunking:exploring adipocyte caveolae[J].Journal of Lipid Research,2007,48(10):2103-2111.  

[38] DING Y B,WU Y B,ZENG R,et al.Proteomic profiling of lipid droplet-associated proteins in primary adipocytes of normal and obese mouse[J].Acta Biochimica et Biophysica Sinica,2012,44(5):394-406.  
文章导航

/