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

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

Chinese Journal of Animal Nutrition >

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

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

SPECIAL ECONOMIC ANIMAL NUTRITION AND FEED

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

Expand

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

Fold

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

Cite this article

TAN Xiuwen , YOU Wei , LIU Xiaomu , JIN Qing , WEI Chen , WAN Fachun , ZHANG Xianglun . Isolation, Culturing and Identification of Beef Cattle Muscle Cells and Establishment of Its Oxidative Stress Model[J]. Chinese Journal of Animal Nutrition, 2020 , 32(5) : 2369 -2378 . DOI: 10.3969/j.issn.1006-267x.2020.05.046

References

[1] ARCHILE-CONTRERAS A C,PURSLOW P P.Oxidative stress may affect meat quality by interfering with collagen turnover by muscle fibroblasts[J].Food Research International,2011,44(2):582-588.

[2] 崔艳军.热应激和氧化应激对肥育猪骨骼肌代谢的影响及硫辛酸的调控作用[D].博士学位论文.北京:中国农业科学院,2016.
[3] CHEN X,ZHANG Q,CHENG Q,et al.Protective effect of salidroside against H₂O₂-induced cell apoptosis in primary culture of rat hippocampal neurons[J].Molecular and Cellular Biochemistry,2009,332(1/2):85-93.
[4] STRANDBERG Y,GRAY C,VUOCOLO T,et al.Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells[J].Cytokine,2005,31(1):72-86.

[5] FERRARI M,SCALVINI A,LOSIO M N,et al.Establishment and characterization of two new pig cell lines for use in virological diagnostic laboratories[J].Journal of Virological Methods,2003,107(2):205-212.

[6] LIN X J,JIANG S Q,JIANG Z Y,et al.Effects of equol on H₂O₂-induced oxidative stress in primary chicken intestinal epithelial cells[J].Poultry Science,2016,95(6):1380-1386.

[7] LIU X M,LIU G F,TAN X W,et al.Gene expression profiling of SIRT1,FoxO1,and PPARγ in backfat tissues and subcutaneous adipocytes of Lilu bulls[J].Meat Science,2014,96(2):704-711.

[8] LIU X M,ZHAO H B,JIN Q,et al.Resveratrol induces apoptosis and inhibits adipogenesis by stimulating the SIRT1-AMPKα-FOXO1 signalling pathway in bovine intramuscular adipocytes[J].Molecular and Cellular Biochemistry,2018,439(1/2):213-223.
[9] 何芳婷,陈嘉熠,徐佳伊,等.氧化应激细胞模型建立的研究进展[J].食品工业科技,2019,40(7):341-345.
[10] PASZTI-GERE E,CSIBRIK-NEMETH E,SZEKER K,et al.Acute oxidative stress affects IL-8 and TNF-α expression in IPEC-J2 porcine epithelial cells[J].Inflammation,2012,35(3):994-1004.

[11] 郑延松,李源,张珊红,等.用低浓度过氧化氢建立心肌细胞氧化损伤模型[J].第四军医大学学报,2001,22(20):1849-1851.
[12] 金鹿.维生素A对奶牛乳腺硒蛋白合成及抗氧化功能影响机理的研究[D].博士学位论文.呼和浩特:内蒙古农业大学,2014.
[13] 卢靖坤.MSTN蛋白结构变异对牛肌肉生长的影响[D].硕士学位论文,呼和浩特:内蒙古大学,2018.
[14] HINDI L,MCMILLAN J D,AFROZE D,et al.Isolation,culturing,and differentiation of primary myoblasts from skeletal muscle of adult mice[J].Bio-Protocol,2017,7(9):e2248.
[15] MITCHELL K J,PANNÉREC A,CADOT B,et al.Identification and characterization of a non-satellite cell muscle resident progenitor during postnatal development[J].Nature Cell Biology,2010,12(3):257-266.

[16] FACCHINETTI F,FUREGATO S,TERRAZZINO S,et al.H₂O₂ induces upregulation of Fas and Fas ligand expression in NGF-differentiated PC12 cells:modulation by cAMP[J].Journal of Neuroscience Research,2002,69(2):178-188.

[17] 吕晓,朱海鲸,曹晖,等.猪肌肉卫星细胞的分离培养及生物学特性鉴定[J].中国兽医学报,2011,31(10):1480-1484.
[18] 徐蓬,顾晓明.兔骨骼肌卫星细胞的体外培养及生长特性的研究[J].实用口腔医学杂志,2000,16(1):7-9.
[19] GROUNDS M D,GARRETT K L,LAI M C,et al.Identification of skeletal muscle precursor cells in vivo by use of MyoD1 and myogenin probes[J].Cell and Tissue Research,1992,267(1):99-104.

[20] SEALE P,SABOURIN L A,GIRGIS-GABARDO A,et al.Pax7 is required for the specification of myogenic satellite cells[J].Cell,2000,102(6):777-786.

[21] ZHU L H,ZHAO K L,CHEN X L,et al.Impact of weaning and an antioxidant blend on intestinal barrier function and antioxidant status in pigs[J].Journal of Animal Science,2012,90(8):2581-2589.

[22] 蔡旋,王静娴,陈小连,等.肠道上皮氧化应激细胞模型的研究进展[J].畜牧兽医学报,2014,45(3):337-346.
[23] CAI X,CHEN X L,WANG X C,et al.Pre-protective effect of lipoic acid on injury induced by H₂O₂ in IPEC-J2 cells[J].Molecular and Cellular Biochemistry,2013,378(1/2):73-81.
[24] JIN X L,WANG K,LIU H Y,et al.Protection of bovine mammary epithelial cells from hydrogen peroxide-induced oxidative cell damage by resveratrol[J].Oxidative Medicine and Cellular Longevity,2016,2016:2572175.
[25] 戴青里,孙贵龙,闫斌,等.过氧化氢诱导HUVECs氧化应激模型的构建[J].昆明医科大学学报,2018,39(4):34-39.
[26] ALFONSO-PRIETO M,BIARNÉS X,VIDOSSICH P,et al.The molecular mechanism of the catalase reaction[J].Journal of the American Chemical Society,2009,131(33):11751-11761.

[27] VALKO M,LEIBFRITZ D,MONCOL J,et al.Free radicals and antioxidants in normal physiological functions and human disease[J].The International Journal of Biochemistry & Cell Biology,2007,39(1):44-84.

[28] PIAO M J,KANG K A,ZHANG R,et al.Hyperoside prevents oxidative damage induced by hydrogen peroxide in lung fibroblast cells via an antioxidant effect[J].Biochimica et Biophysica Acta:General Subjects,2008,1780(12):1448-1457.

[29] ROSEN J E,PRAHALAD A K,WILLIAMS G M.8-Oxodeoxyguanosine formation in the DNA of cultured cells after exposure to H₂O₂ alone or with UVB or UVA irradiation[J].Photochemistry and Photobiology,1996,64(1):117-122.

[30] LI J,SUN J H,LI B,et al.Astaxanthin protects ARPE-19 cells against oxidative stress injury induced by hydrogen peroxide[J].Biotechnology & Biotechnological Equipment,2018,32(5):1277-1284.

[31] YOUNG D Y,FAN M Z,MINE Y.Egg yolk peptides up-regulate glutathione synthesis and antioxidant enzyme activities in a porcine model of intestinal oxidative stress[J].Journal of Agricultural and Food Chemistry,2010,58(13):7624-7633.

[32] QUINCOZES-SANTOS A,BOBERMIN L D,LATINI A,et al.Resveratrol protects C6 astrocyte cell line against hydrogen peroxide-induced oxidative stress through heme oxygenase 1[J].PLoS One,2013,8(5):e64372.
[33] 金晓露.白藜芦醇抵御奶牛乳腺上皮细胞氧化应激的作用机制研究[D].博士学位论文,杭州:浙江大学,2016.
[34] DEY S,SIDOR A,O'ROURKE B.Compartment-specific control of reactive oxygen species scavenging by antioxidant pathway enzymes[J].Journal of Biological Chemistry,2016,291(21):11185-11197.

Options

Outlines

模态框（Modal）标题

Abstract

Cite this article

References