山羊胎儿肌肉干细胞的分离培养与成肌诱导分化

doi:10.3864/j.issn.0578-1752.2018.08.016

Abstract

Abstract: 【Objective】 To establish the method for isolation, culture, identification and myogenic differentiation of fetal muscle stem cells from Anhuai goat in vitro, and to provide experimental materials for further research on the molecular mechanism of goat muscle stem cell proliferation and differentiation. 【Method】 In this study, the goat fetal longissimus muscle tissue was selected and cut into meat emulsion with ophthalmology, digested with 0.1% type I collagenase for 40 min and then digested with 0.25% trypsin for 15 min. The isolated cells were cultured in growth medium (20% FBS + 80% DMEM / F12 + Penicillin) in a 37℃, 5% CO2 incubator. After culturing for 2h, the cells were purified by differential adherent technique. After 2h, the cells were purified again. The cells were subcultured when they reached 70% density. 30min adherent method was used for further purification with each subculture of muscle stem cells until the sixth passages. Muscle stem cell marker genes Pax7 and MyoD1 were detected with the purified cells for identification. When muscle stem cells grew to a density of about 70%, the growth medium was displaced with differentiation medium (2% FBS + 98% DMEM / F12 + Penicillin)for myoblasts induction and the morphology of the cells was monitored . One day after the induction of the cells, the marker protein of muscle stem cells Myog was detected. In addition, total RNAs of cells induced at 0, 1, 3, 5, and 7 days were separately extracted and their relative expression amounts of MyoD1 and Myog genes were measured by qPCR. 【Result】 Isolated cells showed adherent growth, and their morphology tended to be long spindle after stabilization. Pax7 and MyoD1 expression were detected in the 6th passage cells by immunofluorescence. After induction by differentiated medium, the cells started to differentiate and fuse with each other into myotubes with a certain directionality as the induction prolonged. Myog protein was detected by immunofluorescence assay. Differentiation marker genes MyoD1 and Myog were detected by qPCR. Expression of MyoD1 could be detected in the first day of induction and maintained until the 3rd day, and its level began to decline from the 5th day but still significantly higher than the proliferative phase. A similar dynamic was observed with the relative expression level of Myog in the differentiating cells.【Conclusion】In this experiment, the fetal muscle stem cells of Anhuai goat was obtained with high purity, which showed good myogenic potential after induction. The results provide material for further research on the mechanisms of myogenic differentiation of muscle stem cells.

Key words: goat fetus, muscle stem cells, isolation culture, identification, myogenic differentiation

SUI MengHua, ZHENG Qi, WU Hao, DING JianPing, LIU Yong, LI WenYong, CHU MingXing, ZHANG ZiJun, LING YingHui. Isolation, Culture and Myogenic Differentiation of Muscle Stem Cells in Goat Fetal[J].Scientia Agricultura Sinica, 2018, 51(8): 1590-1597.

References

[1] STANTZOU A, SCHIRWIS E, SWIST S, ALONSO M S, POLYDOROU I, ZARROUKI F, MOUISEL E, BELEY C, JULIEN A, LE G F, GARCIA L, COLNOT C, BIRCHMEIER C, BRAUN T, SCHUELKE M, RELAIX F, AMTHOR H. BMP signaling regulates satellite cell dependent postnatalmuscle growth. Development, 2017, 114(15): 2737.

[2] KOKABU S, NAKATOMI C, MATSUBARA T, ONO Y, ADSION W N, LOWERY J W, URATA M, HUDNALL A M, HITOMI S, NAKATOMI M, SATO T, OSAWA K, YODA T, ROSEN V, JIMI E. The transcriptional co-repressor TLE3 regulates myogenic differentiation by repressing the activity of the MyoD transcription factor. The Journal of biological chemistry, 2017, 292: jbc.M116.774570.

[3] RUDNICKI M A, LE G F, MCKINNELL I, KUANG S. The molecular regulation of muscle stem cell function. Cold Spring Harbor Symposia on Quantitative Biology, 2008, 73:323-331.

[4] ZHANG W W, SUN X F, TONG H L, WANG Y H, LI S F, YAN Y Q, LI G P. Effect of differentiation on microRNA expression in bovine skeletal muscle satellite cells by deep sequencing. Cellular & Molecular Biology Letters, 2016 , 21(1):8.

[5] SUN C, DE M V, MOHAMED A, ORTUSTE QUIROGA H P, ARCIA M A, TREMBLAY A M, VON K A, COLLIE D E, VARGESSON N, MATALLANAS D, WACKERHAGE H, ZAMMIT P S. Common and distinctive functions of the hippo effectors taz and yap in skeletal muscle stem cell function. Stem Cells, 2017, 35(8):1958.

[6] DAI Y, ZHANG W R, WANG Y M, LIU X F, LI X, DING X B, GUO H. MicroRNA-128 regulates the proliferation and differentiation of bovine skeletal muscle satellite cells by repressing Sp1. Molecular & Cellular Biochemistry, 2016, 414(1-2):37.

[7] MENDIAS C L. Fibroblasts take the center stage in human skeletal muscle regeneration. The Journal of Physiology, 2017, 595(15):5005.

[8] ZHANG W R, ZHANG H N, WANG Y M, DAI Y, LIU X F, LI X, DING X B, GUO H. MiR-143 regulates proliferation and differentiation of bovine skeletal muscle satellite cells by targeting IGFBP5. Vitro Cellular & Developmental Biology Animal, 2017, 53(3): 265.

[9] GOKULAKRISHNAN G, CHANG X, FLEISCHNANN R, FIOROTTO M L. Precocious glucocorticoid exposure reduces skeletal muscle satellite cells in the fetal rat. Journal of Endocrinology, 2017, 232(3):561-572.

[10] TIM S, NWDERVEEN J P, MCKAY B R, SOPHIE J, LEX B V, GIANNI P. Satellite cells in human skeletal muscle plasticity. Frontiers in Physiology, 2015, 6: 283.

[11] LI B J, LI P H, HUANG R H, SUN W X, WANG H, LI Q F, CHEN J, WU W J, LIU H L. Isolation, culture and identification of porcine skeletal muscle satellite cells. Asian-Australasian Journal of Animal Sciences, 2015, 28(8):1171-1177.

[12] SOUSA V P, García-Prat L, SERRANO A L, PERDIGUERO E, MUNOZ-CANOVES P. Muscle stem cell aging: regulation and rejuvenation. Trends in Endocrinology & Metabolism, 2015, 26:287-296.

[13] WANG Y M, DING X B, DAI Y, LIU X F, GUO H, ZHANG Y. Identification and bioinformatics analysis of miRNAs involved in bovine skeletal muscle satellite cell myogenic differentiation. Molecular & Cellular Biochemistry, 2015, 404(1-2):113.

[14] BRAGA M, SIMMONS Z, NORRIS K C, FERRINI M G, ARTAZA J N. Vitamin D induces myogenic differentiation in skeletal muscle derived stem cells. Endocrine Connections, 2017, 6(3):139-150.

[15] PINI V, MORGAN J E, MUNTONI F, O'NEILL H C. Genome editing and muscle stem cells as a therapeutic tool for muscular dystrophies. Current Stem Cell Reports, 2017: 1-12.

[16] PARTRIDGE T A, GROUNDS M, SLOSPER J C. Evidence of fusion between host and donor myoblasts in skeletal muscle grafts. Nature, 1978, 273(5660):306-308.

[17] DUMONT N A, BENTZINGER C F, SINCENNES M, RUDNICKI M A. Satellite cells and skeletal muscle regeneration. Comprehensive Physiology, 2015, 5(3):1027.

[18] PERSSON P B. Skeletal muscle satellite cells as myogenic progenitors for muscle homeostasis, growth, regeneration and repair. Acta Physiologica, 2015, 213(3):537-538.

[19] SOUSA V P, GARCIA P L, SERRANO A L, PERDIGUERO E, MUNOZ C P. Muscle stem cell aging: regulation and rejuvenation. Trends in Endocrinology & Metabolism, 2015, 26:287-296.

[20] MONTOYA-FLORES D, MORA O, TAMARIZ E, GONZALEZ- DAVALOS L, GONZALEZ-GALLARDO A, ANTARAMIAN A, SHIMADA A, VARELA-ECHAVARRIA A, ROMANO-MUNOZ J L. Ghrelin stimulates myogenic differentiation in a mouse muscle satellite cellline and in primary cultures of bovine myoblasts. Journal of Animal Physiology and Animal Nutrition, 2012, 96(4):725-738.

[21] 李方华, 侯玲玲, 马月辉, 庞全海, 关伟军. 北京油鸡骨骼肌卫星细胞的分离、培养、鉴定及成肌诱导分化的研究. 中国农业科学, 2010, 43(22):4725-4731.

LI F H, HOU L L, MA Y H, PANG Q H, GUAN W J. Study on isolation, culture, identification and differentiation of skeletal muscle satellite cells in Beijing oil chicken. Chinese Journal of Agricultural Sciences, 2010, 43 (22): 4725-4731. (in Chinese)

[22] SHEFER G, YABLONKAREUVENI Z. Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells. Methods in Molecular Biology, 2005, 946(290):281-304.

[23] YAMANOUCHI K, HOSOYAMA T, MURAKAMI Y, NAKANO S, NISHIHARA M. Satellite cell differentiation in goat skeletal muscle single fiber culture. Journal of Reproduction and Development, 2009, 55(3):252-255.

[24] SEALE P, RUDNICKI M A. A new look at the origin, function, and "stem-cell" status of muscle satellite cells. Developmental Biology, 2000, 218(2):115-124.

[25] 何波, 郑嵘, 熊远著,胡春艳. 新生猪骨骼肌卫星细胞的培养鉴定及生物学特性. 畜牧兽医学报, 2006, 37(6):555-559.

HE B, ZHENG R, XIONG Y Z, HU C Y. Culture and identification of skeletal muscle satellite cells of newborn pigs and their biological characteristics. Journal of Animal Husbandry and Veterinary Medicine, 2006, 37 (6): 555-559. (in Chinese)

[26] MAESNER C C, ALMADA A E, WAGERS A J. Established cell surface markers efficiently isolate highly overlapping populations of skeletal muscle satellite cells by fluorescence-activated cell sorting. Skeletal Muscle, 2016, 6(1):35.

[27] 陈岩, 王琨, 朱大海. 鸡骨骼肌卫星细胞的分离培养、鉴定及生物学特性研究. 遗传, 2006, 28(3):257-260.

CHEN Y, WANG K, ZHU D H. Isolation, culture and identification of chicken skeletal muscle satellite cells and their biological characteristics. Genetic, 2006, 28(3):257-260. (in Chinese)

[28] 李俊涛，赵薇，李丹丹，冯静，巴贵，宋天增，张红平. miR-101a靶向EZH2促进山羊骨骼肌卫星细胞的分化. 遗传, 2017, 39(9): 828-836.

LI J T, ZHAO W, LI D D, FENG J, BA G, SONG T Z, ZHANG H P. EZH2 targets miR-101a in goat skeletal muscle satellite cells differentiation. Genetic, 2017, 39(9): 828-836. (in Chinese)

[29] 吴海青. mTOR信号通路对山羊骨骼肌卫星细胞增殖及分化的影响[D]. 呼和浩特: 内蒙古大学，2015.

WU H Q. The effects of mammalian target of rapamycin signaling pathway on proliferation and differentiation of goat skeletal muscle satellite cells [D]. Huhhot: Mongolian university, 2015. (in Chinese)

[30] YIN H, PRICE F, RUDNICKI M A. Satellite cells and the muscle stem cell niche. Physiological Reviews, 2013, 93(1):23.

[31] XU X, JI S, LI W, YI B, LI H X, ZHANG H F, MA W P. LncRNA H19 promotes the differentiation of bovine skeletal muscle satellite cells by suppressing Sirt1/FoxO1. Cellular & Molecular Biology Letters, 2017, 22(1):10.

[32] GRIGER J, SCHNEIDER R, LAHMANN I, SCHOWEL V, KELLER C, SPULER S, NAZARE M, BIRCHMEIER C. Loss of Ptpn11 (Shp2) drives satellite cells into quiescence. Elife, 2017, 6.

[33] LAUMONIER T, BERMONT F, HOFFEYER P, KINDLER V, MENETREY J. Human myogenic reserve cells are quiescent stem cells that contribute to muscle regeneration after intramuscular transplantation in immunodeficient mice. Scientific Reports, 2017, 14;7(1):3462.

[34] LILJA K C, ZHANG N, MAGLI A, GUNDUZ V, BOWMAN C J, ARPKE R W, DARABI R, KYBA M, PERLINGEIRO R, DYNLACHT B D. Pax7 remodels the chromatin landscape in skeletal muscle stem cells. PLoS One, 2017, 12(4):e0176190.

[35] STAVROULA T, DELLAG P A, RUSSELL A P. Skeletal muscle satellite cells, mitochondria, and microRNAs: their involvement in the pathogenesis of ALS. Frontiers in Physiology, 2016, 7: 403.

[36] 刘月光, 史新娥, 沈清武,袁媛,杨秋梅,高晓娟,陈宗正,杨公社. 利用单根肌纤维法分离和培养猪骨骼肌卫星细胞及其成肌诱导分化. 农业生物技术学报, 2011, 19(5):856-863.

LIU Y G, SHI X E, SHEN Q W, YUAN Y, YANG Q M, GAO X J, YANG Z Z,YANG G S. Isolation and culture of porcine skeletal muscle satellite cells and their myogenic differentiation by single muscle fiber method. Journal of Agricultural Biotechnology, 2011, 19(5):856-863. (in Chinese)

[37] WU H, REN Y, LI S, WANG W, YUAN J, GUO X, LIU D, CANG M . In vitro culture and induced differentiation of sheep skeletal muscle satellite cells. Cell Biology International, 2012, 36(6):579-587.

[38] MOTOHASHI N, ASAKURA A. Muscle satellite cell heterogeneity and self-renewal. Frontiers in Cell Developmental Biology, 2014, 2(1):1.

Related Articles 15

[1]	SUN YaLing, WANG QingHua, SHU Rui, YUE LiXin, WANG ZhenBao, LI ZhaoXia, GAO LiMin, CHENG Hong, FU ZaiQiu, HUO YuMeng. Production and Characterization of Interspecific Hybrids Between Allium fistulosum and Allium przewalskianum Regel [J]. Scientia Agricultura Sinica, 2026, 59(3): 619-636.
[2]	HE ZhiLin, SUN CuiXia, YUE HongLi, TAN YueXia, ZHANG YaoHai, WANG FuSheng, LIU SiTao, JIANG Dong. Genetic Diversity Analysis and GWAS of Alloocimene Based on Resequencing of Citron, Lemon Germplasm Resources [J]. Scientia Agricultura Sinica, 2026, 59(2): 386-401.
[3]	XU DuoDuo, DU QianQian, ZHAO LiXiang, LI Yan, HUANG Gan, LI YongHua, LU JiuXing. Genome-Wide Analysis of AP2/ERF Transcription Factors in Peony [J]. Scientia Agricultura Sinica, 2025, 58(23): 5031-5045.
[4]	ZHANG ZhiChun, HE XiaoYi, HE ChengJun, GU DongLin, ZHONG Lian, GOU Yan, GAO BiXing, GENG Zhao, LI XiaoHong, CHEN SiKai. Comparative Analysis of Volatile Oil Chemical Constituents from Zanthoxylum bungeanum Seeds in Yanyuan and Hanyuan Based on GC-MS Technology [J]. Scientia Agricultura Sinica, 2025, 58(23): 5057-5070.
[5]	CAI HaiLin, HUANG XinRong, ZENG WeiAi, PENG ZhiXin, ZHAO AJuan, BO ChunBin, XIANG ShiPeng, GAO PengCheng, ZHANG ChengSheng, ZHAO DongLin. Structure and Herbicidal Activity of 3,5-Dimethylorsellinic Acid-Derived Meroterpenoids from Penicillium sclerotiorum HY5 [J]. Scientia Agricultura Sinica, 2025, 58(22): 4673-4687.
[6]	SU XiaoMei, YANG ZongHui, LIU ShuMei, ZHANG ZongJie, LÜ HongJun, HOU LiXia. Development and Application of A KASP Marker-Based Identification System for Tomato Varieties [J]. Scientia Agricultura Sinica, 2025, 58(22): 4746-4756.
[7]	WU ShuYu, HENG YanFang, YU TaiFei, WANG ShiJia, YU SiJia, LI Yuan, HU Zheng, ZHANG Hui, SUN XianJun, LI Liang, JIANG QiYan. Identification of Salt Tolerance in Maize Natural Populations at the Seedling Stage and Analysis of Salt Tolerance-Associated Genes [J]. Scientia Agricultura Sinica, 2025, 58(20): 4085-4099.
[8]	CHEN YongXian, CHEN RuiJiang, DU YiZhi, ZHU JunJie, CHEN WanXia, ZHAO ZiHan, WANG JiChun, DU Kang, ZHANG Kai. Screening and Identification of Drought-Tolerant Sweet Potato Germplasm Resources [J]. Scientia Agricultura Sinica, 2025, 58(2): 214-237.
[9]	PAN Yuan, WANG De, LIU Nan, MENG XiangLong, DAI PengBo, LI Bo, HU TongLe, WANG ShuTong, CAO KeQiang, WANG YaNan. Evaluation of the Effectiveness of Two High-Throughput Sequencing Techniques in Identifying Apple Viruses and Identification of Two Novel Viruses [J]. Scientia Agricultura Sinica, 2025, 58(2): 266-280.
[10]	YI ZeHui, WANG Ying, SONG HuiXia, ZHAO Jing, MAO LiPing. Genome-Wide Identification and Expression Analysis of Peroxiredoxins Gene Family in Asparagus officinalis [J]. Scientia Agricultura Sinica, 2025, 58(18): 3728-3743.
[11]	LI YaYu, WANG XinLiang, ZHOU JinHuan, LI ChuXin, LI JiaXin, TIAN XuBin, SONG Zhen. Construction and Infectivity Identification of Genome-Length cDNA of Citrus Psorosis Virus [J]. Scientia Agricultura Sinica, 2025, 58(17): 3451-3460.
[12]	ZHAO YuMeng, LI GuangXu, DAI QiDong, LIU JiaCheng, ZHAO HaiJuan, ZHANG YuJun, YANG Hua, LIU Shuo. Isolation and Identification of Pathogen of Plum Bacterial Shot Hole and Laboratory Test of Bactericide Toxicity [J]. Scientia Agricultura Sinica, 2025, 58(12): 2358-2370.
[13]	WEI NaiCui, TAO JinBo, YUAN MingYang, ZHANG Yu, KAI MengXiang, QIAO Ling, WU BangBang, HAO YuQiong, ZHENG XingWei, WANG JuanLing, ZHAO JiaJia, ZHENG Jun. Seedling Characterization and Genetic Analysis of Low Phosphorus Tolerance in Shanxi Varieties [J]. Scientia Agricultura Sinica, 2024, 57(5): 831-845.
[14]	CHEN XiHong, CAI Wei, YU Yun, LI Min, WANG NianWu, DU ZhenGuo, SHEN JianGuo, GAO FangLuan. Identification of Tea Plant Viruses in Fujian Province and Establishment of Multiplex PCR Detection Assay [J]. Scientia Agricultura Sinica, 2024, 57(4): 698-710.
[15]	LI XinLei, SUN JiuYing, YANG Cheng, CHENG Ning, WANG KaiYue, WANG HuanHuan, CHENG XueJiao, ZHAO Jian, SUN YingFeng. Isolation, Identification and Genetic Variation of a Three-Lineage Strain Recombined Porcine Reproductive and Respiratory Syndrome Virus [J]. Scientia Agricultura Sinica, 2024, 57(24): 4978-4989.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

Isolation, Culture and Myogenic Differentiation of Muscle Stem Cells in Goat Fetal

RichHTML

PDF