动物营养学报 >

2020 , Vol. 32 >Issue 5: 2369 - 2378

DOI: https://doi.org/10.3969/j.issn.1006-267x.2020.05.046

实验方法与实验动物 EXPERIMENTAL METHOD AND ANIMAL

肉牛肌肉细胞的分离培养和鉴定及其氧化应激模型的建立

  • 谭秀文 ,
  • 游伟 ,
  • 刘晓牧 ,
  • 靳青 ,
  • 魏晨 ,
  • 万发春 ,
  • 张相伦
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  • 山东省农业科学院畜牧兽医研究所, 山东省畜禽疫病防治与繁育重点实验室, 山东省肉牛生产性能测定中心, 山东省畜禽健康养殖工程技术中心, 济南 250100
谭秀文(1979-),女,山东潍坊人,副研究员,博士,从事动物繁殖与胚胎工程研究。E-mail:lafta@sina.com

收稿日期: 2019-10-29

  网络出版日期: 2020-05-15

基金资助

山东省自然科学基金项目（ZR2017BC043）；现代农业（肉牛牦牛）产业技术体系建设专项资金（CARS-37）；山东省农业科学院农业科技创新工程（CXGC2017B02）；山东省农业科学院畜牧兽医研究所资助项目

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Isolation, Culturing and Identification of Beef Cattle Muscle Cells and Establishment of Its Oxidative Stress Model

  • TAN Xiuwen ,
  • YOU Wei ,
  • LIU Xiaomu ,
  • JIN Qing ,
  • WEI Chen ,
  • WAN Fachun ,
  • ZHANG Xianglun
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  • Shandong Provincial Engineering Technology Center of Animal Healthy Breeding, Shandong Provincial Testing Center of Beef Cattle Performance, Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Ji'nan 250100, China

Received date: 2019-10-29

  Online published: 2020-05-15

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摘要

本研究采用组织块法分离培养获得肉牛肌肉细胞并进行鉴定，利用过氧化氢（H2O2）构建氧化应激模型，为深入研究氧化应激对肉牛肌肉生长发育和肉品质的影响机制提供技术支撑。选用体重为252 kg的利鲁（利木赞×鲁西黄牛）杂交公牛背最长肌，利用组织块培养法分离培养肌肉细胞。通过形态学观察、免疫荧光法及流式细胞仪等手段对细胞进行鉴定。采用不同浓度（0、50、100、150、200、250、300、350、400、450 μmol/L）的H2O2处理细胞24 h，通过观察细胞形态、检测细胞存活率及氧化还原状态，筛选适宜作用浓度。然后利用该浓度H2O2处理细胞不同时间（0、0.75、1.5、3、6、12、24 h），通过分析确定适宜作用时间。结果表明：1）分离获得的细胞长势良好，形状细长，呈纺锤体形和梭形。免疫荧光法和流式细胞仪鉴定肌肉细胞的特异性蛋白（生肌决定因子1和配对盒基因7）均得到较高表达。2）视野内细胞数量随H2O2浓度升高逐渐减少，至450 μmol/L时，细胞发生明显死亡现象。细胞存活率随H2O2浓度的升高显著降低，与0浓度H202处理相比，300 μmol/L时降至65.4%（P<0.05），450 μmol/L时仅为6.7%（P<0.05）。细胞活性氧（ROS）水平先略微降低后逐渐升高（P<0.05），超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性先升高后逐渐降低（P<0.05），丙二醛（MDA）和蛋白质羰基（PC）含量逐渐升高（P<0.05），8-羟基脱氧鸟苷（8-OHdG）含量先升高后降低（P<0.05）。3）细胞存活率随300 μmol/L H2O2处理时间的延长逐渐降低，与0 h相比，6 h降至61.2%（P<0.05），12 h降至47.4%（P<0.05），但在24 h时略有升高（P<0.05）。随着处理时间的延长，细胞ROS水平先升高后逐渐降低（P<0.05），SOD和CAT活性先升高后降低（P<0.05），MDA、PC和8-OHdG含量均逐渐升高（P<0.05）。综上所述，本研究通过组织块培养法分离培养获得较高纯度的肉牛肌肉细胞，并成功构建氧化应激模型，H2O2的适宜作用浓度和时间分别为300 μmol/L和6 h。

关键词： 肉牛; 肌肉细胞; 细胞培养; 细胞鉴定; 过氧化氢; 氧化应激模型

本文引用格式

谭秀文 , 游伟 , 刘晓牧 , 靳青 , 魏晨 , 万发春 , 张相伦 . 肉牛肌肉细胞的分离培养和鉴定及其氧化应激模型的建立[J]. 动物营养学报, 2020 , 32(5) : 2369 -2378 . DOI: 10.3969/j.issn.1006-267x.2020.05.046

Abstract

This study was conducted to isolate the beef cattle muscle cells using tissue cultivation method followed by cell identification. Oxidative stress model of beef cattle muscle cells was further established by hydrogen peroxide (H2O2), which could provide technical support for the mechanism research of oxidative stress on muscle tissue growth and meat quality. Longissimus dorsi tissue from a Lilu (Limousin×Luxi cattle, 252 kg body weight) crossbred bull was collected and muscle cells were isolated using tissue cultivation method. Identification of the cells was conducted by morphological observation, immunofluorescence and flow cytometry method. The optimal concentration of H2O2 for cell oxidative stress model was determined by analyzing cell morphology, viability and redox status of the cells treated with different concentrations of H2O2 (0, 50, 100, 150, 200, 250, 300, 350, 400 and 450 μmol/L) for 24 h. Then, the appropriate treatment time (0, 0.75, 1.5, 3, 6, 12 and 24 h) of H2O2 was further determined based on the optimal concentration of H2O2. The results showed as follows:1) cells grew well, which were slender, spindle-type and spindle-like. The specific protein of muscle cell (myogenic determination factor 1 and paired box protein 7) were positively detected by immunofluorescence and flow cytometry method. 2) The quantity of cells in the field of microscope was gradually decreased with the increase concentration of H2O2. Cell death was clearly observed when the concentration of H2O2 was 450 μmol/L. Cell viability was significantly decreased with the increase concentration of H2O2 and the viability reduced to 65.4% or 6.7% when cells incubated with 300 or 450 μmol/L H2O2 compared with 0 concentration of H2O2 treatment, respectively (P<0.05). With the increase concentration of H2O2, reactive oxide species (ROS) levels initially decreased then significantly increased (P<0.05), activities of superoxide dismutase (SOD) and catalase (CAT) initially increased then decreased (P<0.05), contents of malonaldehyde (MDA) and protein carbonyl (PC) gradually increased (P<0.05), and 8-hydroxy-2 deoxyguanosine (8-OHdG) content initially increased then decreased (P<0.05) of the cells. 3) The cell viability was decreased when cells incubated with 300 μmol/L H2O2 for different time, and the viability reduced to 61.2% or 47.4% at 6 or 12 h incubation compared with 0 h, respectively (P<0.05). However, the cell viability was slightly increased at 24 h incubation (P<0.05). Additionally, the ROS levels significantly increased and then gradually decreased (P<0.05), activities of SOD and CAT initially increased then decreased (P<0.05), and contents of MDA, PC and 8-OHdG all gradually increased (P<0.05) along with the extension of incubation time. In conclusion, beef cattle muscle cells with high purity are successfully isolated by tissue cultivation method in the study. The condition that 300 μmol/L H2O2 reacted for 6 h is optimal for establishment of oxidative stress model of beef cattle muscle cells.

Key words： beef cattle; muscle cells; cell culture; cell identification; hydrogen peroxide; oxidative stress cell model

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