作 者：
闫峻;马衍旋;穆淑琴;唐湘方;郑梓;李宁;刘陈龙;李千军;孙超
单 位：
天津大学化工学院;西北农林科技大学动物科技学院;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;天津市农业科学院天津市畜牧兽医研究所
关键词：
猪;骨骼肌;肌内脂滴;超速离心;分离纯化
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PM SF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。
译 名：
Isolation and Purification of Intramuscular Lipid Droplets in Porcine Skeletal Muscle
作 者：
YAN Jun;MA Yanxuan;MU Shuqin;TANG Xiangfang;ZHENG Zi;LI Ning;LIU Chenlong;LI Qianjun;SUN Chao;Tianjin Institute of Animal Husbandry and Veterinary Medicine,Tianjin Academy of Agricultural Sciences;Institute of Chemical Technology,Tianjin University;College of Animal Science and Technology,Northwest A& F University;Institute of Animal Sciences of Chinese Academy of Agricultural Sciences State Key Laboratory of Animal Nutrition;
单 位：
YAN Jun%MA Yanxuan%MU Shuqin%TANG Xiangfang%ZHENG Zi%LI Ning%LIU Chenlong%LI Qianjun%SUN Chao%Tianjin Institute of Animal Husbandry and Veterinary Medicine,Tianjin Academy of Agricultural Sciences%Institute of Chemical Technology,Tianjin University%College of Animal Science and Technology,Northwest A& F University%Institute of Animal Sciences of Chinese Academy of Agricultural Sciences State Key Laboratory of Animal Nutrition
关键词：
porcine;;skeletal muscle;;intramuscular lipid droplets;;ultracentrifugation;;isolation and purification
摘 要：
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.
