鹅骨骼肌卫星细胞的分离培养与鉴定

doi:10.11843/j.issn.0366-6964.2023.10.017

Abstract

Abstract: The purpose of the study was to establish methods for isolation, culture, purification, and identification of goose skeletal muscle satellite cells in vitro. The healthy 10-week-old Xupu geese was selected as experimental materials. Satellite cells were isolated from the muscle with the action of Dispase II and collagenase II. Satellite cells were purified by differential adhesion to the culture dish. During in vitro culture, the complete DMEM/F12 growth medium containing 20% FBS and supplemented with bFGF could better maintain the differentiation potential of satellite cells, and the culture dishes involved in the culture process needed to be coated with matrigel specially. After the formation of mature myotubes, the expression of myosin heavy chain (MyHC) was detected by immunofluorescence and Western blotting. The relative expression levels of Pax7, MyoD1 and MyoG before (growth medium, GM) and after differentiation (differentiation medium, DM) were detected by qRT-PCR. There were 3 replicates in each group before and after differentiation. The results showed that the newly isolated satellite cells were small in size and strong in refraction. After adhering to the wall, they had a spindle-shaped structure with needle-like ends and puffed up in the middle. The cells were identified as Pax7 positive by immunofluorescence. Mature multinucleated myotubes could be formed after induction of differentiation. MyHC was positive by immunofluorescence detection. The statistical differentiation index was as high as 78% (P<0.01). The marker genes Pax7, MyoD1, and MyoG were detected positive by qRT-PCR. The relative expression of Pax7 gene before differentiation was 2.22 times (P<0.01) that after differentiation, while the relative expression of marker genes MyoD1 and MyoG after differentiation were 1.90 (P<0.01) and 44.22 times (P<0.01) that before differentiation, respectively. Western blotting results showed that the expression level of MyHC protein was significantly increased after differentiation. In this study, a method for the isolation and culture of goose skeletal muscle satellite cells by collagenase II and dispase II was established. The culture system can enable cells to maintain huge differentiation potential during the in vitro culture. The study results provide a good cell model for the research of the growth and development molecular regulation mechanism of goose muscle tissue.

Key words: goose, skeletal muscle satellite cells, isolation, culture, identification

CLC Number:

S835

ZHANG Li, XU Jialong, HUANG Jinyu, XU Ziyue, LEI Xinnuo, LU Huipeng, ZHU Rui, SUN Weixiang, CAO Haiyue, WANG Anping, ZHU Shanyuan. Isolation, Culture, and Identification of Skeletal Muscle Satellite Cells in Goose[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4186-4195.

References 40

[1]	罗庆斌, 何大乾, 尹荣楷, 等.我国养鹅业现状及发展趋势[J].中国家禽, 2006, 28(4):1-4.LUO Q B, HE D Q, YIN R K, et al.Current situation and development trends on goose industry in China[J].China Poultry, 2006, 28(4):1-4.(in Chinese)
[2]	SONG T, SADAYAPPAN S.Featured characteristics and pivotal roles of satellite cells in skeletal muscle regeneration[J].J Muscle Res Cell Motil, 2020, 41(4):341-353.
[3]	SCHMIDT M, SCHVLER S C, HVTTNER S S, et al.Adult stem cells at work:regenerating skeletal muscle[J].Cell Mol Life Sci, 2019, 76(13):2559-2570.
[4]	MAURO A.Satellite cell of skeletal muscle fibers[J].J Biophys Biochem Cytol, 1961, 9(2):493-495.
[5]	BISCHOFF R.Enzymatic liberation of myogenic cells from adult rat muscle[J].Anat Rec, 1974, 180(4):645-661.
[6]	BLAU H M, WEBSTER C.Isolation and characterization of human muscle cells[J].Proc Natl Acad Sci U S A, 1981, 78(9):5623-5627.
[7]	LU S H, WEI C F, YANG A H, et al.Isolation and characterization of human muscle-derived cells[J].Urology, 2009, 74(2):440-445.
[8]	DING S J, SWENNEN G N M, MESSMER T, et al.Maintaining bovine satellite cells stemness through p38 pathway[J].Sci Rep, 2018, 8(1):10808.
[9]	DODSON M V, MARTIN E L, BRANNON M A, et al.Optimization of bovine satellite cell-derived myotube formation in vitro[J].Tissue Cell, 1987, 19(2):159-166.
[10]	洪倩倩, 郭宏, 高树新, 等.过表达MyBPC1对牛骨骼肌卫星细胞增殖分化的影响[J].畜牧兽医学报, 2021, 52(12):3439-3448.HONG Q Q, GUO H, GAO S X, et al.Effect of overexpression of MyBPC1 on proliferation and differentiation of bovine skeletal muscle satellite cells[J].Acta Veterinaria et Zootechnica Sinica, 2021, 52(12):3439-3448.(in Chinese)
[11]	薛科, 王林杰, 陈利, 等.高糖诱导山羊骨骼肌卫星细胞成脂分化过程中相关基因表达的变化[J].畜牧兽医学报, 2014, 45(5):706-713.XUE K, WANG L J, CHEN L, et al.Adipogenic-related gene expressions in goat skeletal muscle satellite cells treated with high concentration glucose[J].Acta Veterinaria et Zootechnica Sinica, 2014, 45(5):706-713.(in Chinese)
[12]	ZHAO J, ZHAO X Y, SHEN X X, et al.CircCCDC91 regulates chicken skeletal muscle development by sponging miR-15 family via activating IGF1-PI3K/AKT signaling pathway[J].Poult Sci, 2022, 101(5):101803.
[13]	WILSCHUT K J, HAAGSMAN H P, ROELEN B A.Extracellular matrix components direct porcine muscle stem cell behavior[J].Exp Cell Res, 2010, 316(3):341-352.
[14]	DOUMIT M E, MERKEL R A.Conditions for isolation and culture of porcine myogenic satellite cells[J].Tissue Cell, 1992, 24(2):253-262.
[15]	METZGER K, TUCHSCHERER A, PALIN M F, et al.Establishment and validation of cell pools using primary muscle cells derived from satellite cells of pig skeletal muscle[J].In Vitro Cell Dev Biol Anim, 2020, 56(3):193-199.
[16]	WANG H, HE K, ZENG X H, et al.Isolation and identification of goose skeletal muscle satellite cells and preliminary study on the function of C1q and tumor necrosis factor-related protein 3 gene[J].Anim Biosci, 2021, 34(6):1078-1087.
[17]	LE GRAND F, GRIFONE R, MOURIKIS P, et al.Six1 regulates stem cell repair potential and self-renewal during skeletal muscle regeneration[J].J Cell Biol, 2012, 198(5):815-832.
[18]	ROSENBLATT J D, LUNT A I, PARRY D J, et al.Culturing satellite cells from living single muscle fiber explants[J].In Vitro Cell Dev Biol Anim, 1995, 31(10):773-779.
[19]	BILLESKOV T B, JENSEN J B, JESSEN N, et al.Fluorescence-activated cell sorting and phenotypic characterization of human fibro-adipogenic progenitors[J].STAR Protoc, 2023, 4(1):102008.
[20]	LIU L, CHEUNG T H, CHARVILLE G W, et al.Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting[J]. Nat Protoc, 2015, 10(10):1612-1624.
[21]	PAKULA A, SPINAZZOLA J M, GUSSONI E.Purification of myogenic progenitors from human muscle using fluorescence-activated cell sorting (FACS)[M]//RØNNING S B.Myogenesis.New York:Humana Press, 2019:1-15.
[22]	LORETI M, SACCO A.The jam session between muscle stem cells and the extracellular matrix in the tissue microenvironment[J]. NPJ Regen Med, 2022, 7(1):16.
[23]	SU Y, YU Y Y, LIU C C, et al.Fate decision of satellite cell differentiation and self-renewal by miR-31-IL34 axis[J].Cell Death Differ, 2020, 27(3):949-965.
[24]	YOSHIDA T, DELAFONTAINE P.Mechanisms of IGF-1-mediated regulation of skeletal muscle hypertrophy and atrophy[J]. Cells, 2020, 9(9):1970.
[25]	MA N Y, CHEN D L, LEE J H, et al.Piezo1 regulates the regenerative capacity of skeletal muscles via orchestration of stem cell morphological states[J].Sci Adv, 2022, 8(11):eabn0485.
[26]	SCHÄTZLEIN E, BLAESER A.Recent trends in bioartificial muscle engineering and their applications in cultured meat, biorobotic systems and biohybrid implants[J].Commun Biol, 2022, 5(1):737.
[27]	MOISEEVA V, CISNEROS A, SICA V, et al.Senescence atlas reveals an aged-like inflamed niche that blunts muscle regeneration[J]. Nature, 2023, 613(7942):169-178.
[28]	TAGLIETTI V, KEFI K, RIVERA L, et al.Thyroid-stimulating hormone receptor signaling restores skeletal muscle stem cell regeneration in rats with muscular dystrophy[J].Sci Transl Med, 2023, 15(685):eadd5275.
[29]	MUSARÒ A, CAROSIO S.Isolation and culture of satellite cells from mouse skeletal muscle[M]//DI NARDO P, DHINGRA S, SINGLA D K.Adult Stem Cells.New York:Humana Press, 2017:155-167.
[30]	戴巍, 宋瑞龙, 张远浩, 等.鸡骨骼肌卫星细胞的分离培养与鉴定[J].畜牧兽医学报, 2021, 52(3):676-682.DAI W, SONG R L, ZHANG Y H, et al.Isolation, culture and identification of muscle satellite cells of chicken[J].Acta Veterinaria et Zootechnica Sinica, 2021, 52(3):676-682.(in Chinese)
[31]	HE H R, YIN H D, YU X K, et al.PDLIM5 Affects chicken skeletal muscle satellite cell proliferation and differentiation via the p38-mapk pathway[J].Animals (Basel), 2021, 11(4):1016.
[32]	单艳菊, 束婧婷, 宋迟, 等.鸭骨骼肌卫星细胞的分离培养与鉴定[J].江苏农业科学, 2012, 40(12):26-28.SHAN Y J, SHU J T, SONG C, et al.Isolation culture and identification of duck skeletal muscle satellite cells[J].Jiangsu Agricultural Sciences, 2012, 40(12):26-28.(in Chinese)
[33]	林正浩, 王讯, 李晓开, 等.鸽骨骼肌卫星细胞的分离、培养及成肌特性[J].华南农业大学学报, 2019, 40(1):53-58.LIN Z H, WANG X, LI X K, et al.Isolation, identification and biological characteristics of skeletal muscle satellite cells in pigeons[J].Journal of South China Agricultural University, 2019, 40(1):53-58.(in Chinese)
[34]	沈龙仙, 王丽婷, 何珂, 等.褪黑素和烟酰胺单核苷酸对鹅骨骼肌卫星细胞增殖的影响[J].中国农业科学, 2023, 56(2):391-404.SHEN L X, WANG L T, HE K, et al.Effects of melatonin and nicotinamide mononucleotides on proliferation of skeletal muscle satellite cells in goose[J].Scientia Agricultura Sinica, 2023, 56(2):391-404.(in Chinese)
[35]	DANOVIZ M E, YABLONKA-REUVENI Z.Skeletal muscle satellite cells:background and methods for isolation and analysis in a primary culture system[M]//DIMARIO J X.Myogenesis.Totowa:Humana Press, 2012:21-52.
[36]	ZHANG L, WU Y N, LI X, et al.An alternative method for long-term culture of chicken embryonic stem cell in vitro[J].Stem Cells Int, 2018, 2018:2157451.
[37]	SOUSA-VICTOR P, GARCÍA-PRAT L, MUÑOZ-CÁNOVES P.Control of satellite cell function in muscle regeneration and its disruption in ageing[J].Nat Rev Mol Cell Biol, 2022, 23(3):204-226.
[38]	GONZALEZ M L, BUSSE N I, WAITS C M, et al.Satellite cells and their regulation in livestock[J].J Anim Sci, 2020, 98(5):skaa081.
[39]	HERNÁNDEZ-HERNÁNDEZ J M, GARCÍA-GONZÁLEZ E G, BRUN C E, et al.The myogenic regulatory factors, determinants of muscle development, cell identity and regeneration[J].Semin Cell Dev Biol, 2017, 72:10-18.
[40]	CHEN B D, YOU W J, WANG Y Z, et al.The regulatory role of Myomaker and Myomixer-Myomerger-Minion in muscle development and regeneration[J].Cell Mol Life Sci, 2020, 77(8):1551-1569.

Isolation, Culture, and Identification of Skeletal Muscle Satellite Cells in Goose

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 40

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	CHEN Yuetong, LIU Xiaohan, WANG Zhiyang, ZHAO Yuxin, ZHOU Tiezhong, HU Zengjin, ZHU Yue, WANG Shaohui, TIAN Mingxing, DING Siyu, QI Jingjing, YU Shengqing. Isolation, Identification, Pathogenicity and Drug Susceptibility of Mycoplasma gallisepticum from Dead Chicken Embryos in Large-scale Chicken Farms in Guangdong Province [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 290-299.
[2]	YU Xiuju, ZHANG Min'ai, HU Yanjiao, ZHU Zhiwei, WANG Haidong, YANG Lihua, FAN Kuohai. Isolation, Expression and Stability Analysis of Bacteriocins from Bacillus subtilis [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 323-333.
[3]	YANG Heng, LI Zhanhong, SONG Zi'ang, GAO Lin, LI Zhuoran, LIAO Defang, XIAO Lei, LI Huachun. Development and Application of Serotyping Real-time Quantitative RT-PCR Methods for Palyam Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 395-400.
[4]	YU Zhou, YANG Baigao, ZHANG Hang, XU Xi, ZHANG Peipei, FENG Xiaoyi, CAO Jianhua, NIU Yifan, DU Weihua, HAO Haisheng, ZHU Huabin, ABULIZI·Wusiman, ZHAO Xueming. Research Progress of Estrus Markers in Dairy Buffalo [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3623-3630.
[5]	YANG Yue, WANG Rui, GAN Yuan, HAO Fei, XIE Xing, ZHANG Lei, SHAO Guoqing, MENG Qingguo, CHEN Rong, FENG Zhixin. Purification and Identification of SIgA in Porcine Colostrum based on Tandem Affinity Chromatography [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3884-3894.
[6]	FENG Yongzhi, GONG Ting, WU Dongdong, GAO Qi, ZHENG Xiaoyu, ZHANG Guihong, SUN Yankuo. Analysis of Factors Affecting the Infectivity of African Swine Fever Virus on Cultured Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3406-3414.
[7]	LIU Xinhuan, YUN Jialei, MAO Li, LI Jizong, HAO Fei, HE Miaofeng, YANG Leilei, ZHANG Wenwen, CHENG Zilong, SUN Min, LIU Maojun, WANG Shaohui, BAI Juan, LI Wenliang. Isolation, Identification, Virulence Genes and Drug Resistance Analysis of Escherichia coli Isolated from Diarrheal Goat and Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3445-3454.
[8]	CHEN Aolei, HUANG Deru, AN Yafei, LI Shoujun. Animal Intestinal Organoids Culture [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2743-2750.
[9]	QING Enhua, TANG Bincheng, NIU Tian, WANG Junqi, CHEN Zhaoyan, HU Jiwei, HE Hua, LI Liang, WANG Jiwen, HU Shenqiang. Effects of Different Dry Rearing Systems on Histomorphology of Goose Testis and External Genitalia [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2872-2885.
[10]	WANG Jiali, ZHOU Ning, CHEN Xi, YUE Hua, TANG Cheng. Isolation, Identification and Pathogenicity of Canine Adenovirus Type 2 Strain with Natural Deletion in E3 Gene [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2982-2990.
[11]	CHENG Xiangning, ZHANG Yun, HE Jiawei, CHEN Zhengkun, JIN Meilan, DING Honglei, WANG Qinghua. Isolation, Identification and Antifungal Susceptibility Analysis of Candida albicans Isolated from Pigeons [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3002-3011.
[12]	ZHAO Feifei, LI Jie, HAN Ning, XIE Shiting, ZENG Zhenling. Antibacterial Drug Resistance Analysis of Klebsiella pneumoniae Isolated from Slaughterhouse [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3044-3053.
[13]	LIU Hongyi, LUO Tingyu, LI Changwen, YU Haibo, LU Xiaoye, CHEN Hongyan, XIA Changyou, GAO Caixia. Identification of SLA-1 Alleles and Analysis of Molecular Genetic Characteristics in Rongchang Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3064-3077.
[14]	XU Huihao, FENG Xueqian, PIAO Xueling, SHEN Xiaojun, ZHENG Xiaobo, YANG Heng, LIN Jiahao, JIN Yipeng, LIN Degui. Comparative Study on Different Methods of Collection, Isolation, Culture and Identification of Feline Limbal Stem Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3091-3101.
[15]	YUN Jiale, LIU Chang, HUANG Xiaoyu, LIU Qiaoxia, SHI Mingyue, LI Wenxia, NIU Jin, WANG Shouyuan, GAO Pengfei, GUO Xiaohong, LI Bugao, LU Chang, CAO Guoqing. miR-145-5p Inhibits the Proliferation and Differentiation of Porcine Skeletal Muscle Satellite Cells by Targeting IGF1R-Mediated AKT Pathway [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 1893-1904.