共培养体系下牛骨骼肌细胞对脂肪细胞的调控作用研究

doi:10.3864/j.issn.0578-1752.2024.18.015

摘要/Abstract

摘要：

【目的】大理石纹是衡量肉品质的重要指标之一，由牛骨骼肌细胞与牛脂肪细胞共同发育形成。通过最大程度模拟体内肉质形成过程中牛骨骼肌细胞和脂肪细胞互作效应，建立体外两种细胞共培养体系，探究共培养体系下牛骨骼肌细胞分泌和代谢因子对脂肪细胞的调控作用。【方法】采用组织块法和酶消化法分别分离牛脂肪细胞和骨骼肌细胞。利用表型鉴定法和标志基因表达谱鉴定法共同鉴定所分离细胞的纯度和分化潜能，进一步构建牛骨骼肌细胞和牛脂肪细胞条件培养基交换共培养体系以及Transwell共培养体系，通过实时荧光定量PCR（real-time quantitative PCR，qPCR）、EdU染色和油红O染色等方法检测共培养条件下牛骨骼肌细胞分泌和代谢产物对脂肪细胞增殖和分化的影响。【结果】成功构建了两种牛骨骼肌细胞与脂肪细胞的共培养体系。条件培养基交换共培养下，牛脂肪细胞增殖标志基因PCNA、CDK2、CCNE2和CCND1的表达极显著下调（P<0.01）；此外，成脂标志基因FABP4和PPARγ的表达显著下调（P<0.05）；LPL的表达极显著下调（P<0.01）。另一方面，在Transwell共培养条件下，牛脂肪细胞增殖标志基因CCND1的表达显著下调（P<0.05）；PCNA、CDK1和CCNE2的表达极显著下调（P<0.01），且成脂标志基因FABP4的表达显著下调（P<0.05），PPARγ、C/EBPβ和LPL的表达极显著下调（P<0.01）。【结论】牛骨骼肌细胞分泌和代谢产物可通过抑制脂肪细胞增殖标志基因PCNA、CDK1、CDK2、CCNE2和CCND1以及成脂标志基因PPARγ、FABP4、C/EBPβ和LPL的表达抑制牛脂肪细胞的增殖与分化。以上研究结果可为进一步探究肉牛肌内脂肪沉积的分子机制提供技术支撑。

关键词: 牛, 脂肪细胞, 骨骼肌细胞, 共培养, 细胞互作

Abstract:

【Objective】 Marbling is one of the key indicators for meat quality, formed by the joint development of bovine skeletal muscle cells and bovine adipocytes. The purpose of this study was to simulate the interaction effect of bovine skeletal muscle cells and adipocytes in the process of meat formation in vivo to the greatest extent, and to establish two cell co-culture systems in vitro to explore the regulatory effects of bovine skeletal muscle cell secretion and metabolic factors on adipocytes under the co-culture system.【Method】 Bovine adipocytes and skeletal muscle cells were isolated separately using tissue block and enzymatic digestion methods. Cells were identified for purity and differentiation potential by phenotypic identification and gene expression profiling, the conditioned medium exchange co-cultivation systems constructed further, and Transwell co-cultivation systems involving bovine skeletal muscle cells and bovine fat cells was conducted. The impact of secretory and metabolic products from bovine skeletal muscle cells on the proliferation and differentiation of fat cells under co-culture conditions was assessed using techniques, such as real-time quantitative PCR (qPCR), EdU staining, and Oil Red O staining. 【Result】 In this study, two co-culture systems of bovine skeletal muscle cells and adipocytes were successfully constructed. Under the condition of medium exchange co-culture, the expression of bovine fat cell proliferation marker genes, including PCNA, CDK2, CCNE2, and CCND1, was significantly downregulated (P<0.01). Additionally, the expression of adipogenesis marker genes FABP4 and PPARγ was significantly decreased (P<0.05); the expression of LPL was greatly reduced (P<0.01). On the other hand, in the Transwell co-culture system, the expression of the bovine fat cell proliferation marker gene CCND1 was significantly downregulated (P<0.05); the expression of PCNA, CDK1, and CCNE2 was greatly downregulated (P<0.01). Additionally, the expression of the adipogenesis marker gene FABP4 was significantly decreased (P<0.05), while the expression of PPARγ, C/EBPβ, and LPL was extremely significantly decreased (P<0.01). 【Conclusion】 The results of this study indicated that secretory and metabolic products from bovine skeletal muscle cells could suppress the proliferation and differentiation of bovine adipocytes by inhibiting the expression of proliferation marker genes, including PCNA, CDK1, CDK2, CCNE2, and CCND1, as well as lipogenic marker genes, such as PPARγ, FABP4, CEBPβ, and LPL. The results of this study indicated that secretory and metabolic products from bovine skeletal muscle cells could suppress the proliferation and differentiation of bovine adipocytes by inhibiting the expression of proliferation marker genes, such as PCNA, CDK1, CDK2, CCNE2 and CCND1, as well as lipogenic marker genes, such as PPARγ, FABP4, CEBPβ and LPL.

Key words: cattle, adipocyte, skeletal muscle cells, co-culture, cell interactions

杨东梅, 张久盘, 宋雅萍, 宋小雨, 姜超, 马云, 魏大为. 共培养体系下牛骨骼肌细胞对脂肪细胞的调控作用研究[J]. 中国农业科学, 2024, 57(18): 3704-3718.

YANG DongMei, ZHANG JiuPan, SONG YaPing, SONG XiaoYu, JIANG Chao, MA Yun, WEI DaWei. Research on the Regulatory Effects of Bovine Skeletal Muscle Cells on Adipocytes Under Co-Culture Conditions[J]. Scientia Agricultura Sinica, 2024, 57(18): 3704-3718.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2024.18.015

https://www.chinaagrisci.com/CN/Y2024/V57/I18/3704

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参考文献 44

[1]	孙彦泽. 新冠疫情背景下我国肉牛产业现状及发展研究[D]. 大连: 大连海洋大学, 2023.
	SUN Y Z. Study on the current situation and development of beef cattle industry in China under the background of COVID-19 epidemic[D]. Dalian: Dalian Ocean University, 2023. (in Chinese)
[2]	冉桂霞, 李双, 田天, 高淑红. 高档肉牛生产现状及发展方向. 中国畜牧业, 2022(23): 34-35.
	RAN G X, LI S, TIAN T, GAO S H. Present situation and development direction of high-grade beef cattle production. China Animal Industry, 2022(23): 34-35. (in Chinese)
[3]	SONG Y P, WEI D W, RAZA S H A, ZHAO Y A, JIANG C, SONG X Y, WU H, WANG X P, LUORENG Z M, MA Y. Research progress of intramuscular fat formation based on co-culture. Animal Biotechnology, 2023, 34(7): 3216-3236.
[4]	付玉, 张博, 凌遥, 张浩. 骨骼肌生长发育过程及调控研究现状. 中国畜牧兽医, 2021, 48(10): 3565-3574. doi: 10.16431/j.cnki.1671-7236.2021.10.007
	FU Y, ZHANG B, LING Y, ZHANG H. Reviews on process and regulation of skeletal muscle growth and development. China Animal Husbandry & Veterinary Medicine, 2021, 48(10): 3565-3574. (in Chinese)
[5]	束婧婷, 姬改革, 单艳菊, 章明, 肖芹, 屠云洁, 盛中伟, 张笛, 邹剑敏. 基于表达谱芯片挖掘鸡骨骼肌不同类型肌纤维的差异表达基因. 中国农业科学, 2017, 50(14): 2826-2836. doi: 10.3864/j.issn.0578-1752.2017.14.018
	SHU J T, JI G G, SHAN Y J, ZHANG M, XIAO Q, TU Y J, SHENG Z W, ZHANG D, ZOU J M. Analysis of differential expression genes between different myofiber types in chicken skeletal muscle based on gene expression microarray. Scientia Agricultura Sinica, 2017, 50(14): 2826-2836. (in Chinese) doi: 10.3864/j.issn.0578-1752.2017.14.018
[6]	孙加节. 秦川牛肌肉与脂肪组织发育相关miRNA鉴定及miR-10020调控机制解析[D]. 杨凌: 西北农林科技大学, 2015.
	SUN J J. Identification of micrornas from Chinese Qinchuan bovine muscle and adipose tissues and functional analysis of miR-10020 during myogenesis[D]. Yangling: Northwest A & F University, 2015. (in Chinese)
[7]	姜超, 张久盘, 宋雅萍, 宋小雨, 吴昊, 魏大为. FoxO1对牛骨骼肌细胞增殖、凋亡和分化的调控. 中国农业科学, 2024, 57(6): 1191-1203. doi: 10.3864/j.issn.0578-1752.2024.06.013
	JIANG C, ZHANG J P, SONG Y P, SONG X Y, WU H, WEI D W. Study on the role of FoxO1 in regulating the proliferation, apoptosis and differentiation of bovine skeletal muscle cells. Scientia Agricultura Sinica, 2024, 57(6): 1191-1203. (in Chinese) doi: 10.3864/j.issn.0578-1752.2024.06.013
[8]	LI X, FU X, YANG G, DU M. Review: Enhancing intramuscular fat development via targeting fibro-adipogenic progenitor cells in meat animals. Animal, 2020, 14(2): 312-321. doi: 10.1017/S175173111900209X pmid: 31581971
[9]	张潜英. 调控骨骼肌干细胞功能的骨骼肌分泌因子的系统筛选[D]. 北京: 北京协和医学院, 2018.
	ZHANG Q Y. Systematic screening of skeletal muscle secretion factors regulating the function of skeletal muscle stem cells[D]. Beijing: Peking Union Medical College, 2018. (in Chinese)
[10]	CHEN D, LI W F, DU M, CAO B H. Adipogenesis, fibrogenesis and myogenesis related gene expression in longissimus muscle of high and low marbling beef cattle. Livestock Science, 2019, 229: 188-193.
[11]	NGUYEN D V, NGUYEN O C, MALAU-ADULI A E O. Main regulatory factors of marbling level in beef cattle. Veterinary and Animal Science, 2021, 14: 100219.
[12]	ROUDBARI Z, COORT S L, KUTMON M, EIJSSEN L, MELIUS J, SADKOWSKI T, EVELO C T. Identification of biological pathways contributing to marbling in skeletal muscle to improve beef cattle breeding. Frontiers in Genetics, 2020, 10: 1370.
[13]	吕亚宁, 叶文文, 兰旅涛. 牛肌内脂肪沉积影响因素及相关基因研究进展. 中国草食动物科学, 2019, 39(4): 55-57.
	LÜ Y N, YE W W, LAN L T. Research progress on influencing factors and related genes of fat deposition in bovine muscle. China Herbivore Science, 2019, 39(4): 55-57. (in Chinese)
[14]	UTRERA A R, VAN VLECK L D. Heritability estimates for carcass traits of cattle: a review. Genetics and Molecular Research, 2004, 3(3): 380-394. pmid: 15614729
[15]	ZHANG M, LI D H, ZHAI Y H, WANG Z Z, MA X F, ZHANG D Y, LI G X, HAN R L, JIANG R R, LI Z J, KANG X T, SUN G R. The landscape of DNA methylation associated with the transcriptomic network of intramuscular adipocytes generates insight into intramuscular fat deposition in chicken. Frontiers in Cell and Developmental Biology, 2020, 8: 206. doi: 10.3389/fcell.2020.00206 pmid: 32300590
[16]	杜学海. 影响肉牛肌内脂肪沉积因素及相关基因的研究进展. 现代畜牧兽医, 2020(12): 54-57.
	DU X H. Brief introduction to the research progress of factors and related genes influencing intramuscular fat deposition in beef cattle. Modern Journal of Animal Husbandry and Veterinary Medicine, 2020(12): 54-57. (in Chinese)
[17]	HWANG H J, OH M S, LEE D W, KUH H J. Multiplex quantitative analysis of stroma-mediated cancer cell invasion, matrix remodeling, and drug response in a 3D co-culture model of pancreatic tumor spheroids and stellate cells. Journal of Experimental & Clinical Cancer Research, 2019, 38(1): 258.
[18]	FENNEMA E, RIVRON N, ROUWKEMA J, VAN BLITTERSWIJK C, DE BOER J. Spheroid culture as a tool for creating 3D complex tissues. Trends in Biotechnology, 2013, 31(2): 108-115. doi: 10.1016/j.tibtech.2012.12.003 pmid: 23336996
[19]	刘琦, 姚茜, 韦正波, 谢莹. 类器官培养基主要成分的作用机制及潜在功能. 中国组织工程研究, 2021, 25(31): 5072-5078.
	LIU Q, YAO X, WEI Z B, XIE Y. Action mechanism and potential function on main components of organoid culture medium. Chinese Journal of Tissue Engineering Research, 2021, 25(31): 5072-5078. (in Chinese)
[20]	ZHANG Y, GUO W M, WANG M J, HAO C X, LU L, GAO S, ZHANG X L, LI X, CHEN M X, LI P H, JIANG P, LU S B, LIU S Y, GUO Q Y. Co-culture systems-based strategies for articular cartilage tissue engineering. Journal of Cellular Physiology, 2018, 233(3): 1940-1951. doi: 10.1002/jcp.26020 pmid: 28548713
[21]	PANDURANGAN M, HWANG I. Application of cell co-culture system to study fat and muscle cells. Applied Microbiology and Biotechnology, 2014, 98(17): 7359-7364. doi: 10.1007/s00253-014-5935-9 pmid: 25038928
[22]	LU X Q, LIU X F, ZHONG H, ZHANG W, YU S Z, GUAN R F. Progress on three-dimensional cell culture technology and their application. Shengwu Yixue Gongchengxue Zazhi, 2023, 40(3): 602-608.
[23]	杨小倩, 宋爱梅, 宋晖. 间充质干细胞与巨噬细胞的共培养技术. 中国组织工程研究, 2024, 28(31): 5055-5062.
	YANG X Q, SONG A M, SONG H. Co-culture technology of mesenchymal stem cells and macrophages. Chinese Journal of Tissue Engineering Research, 2024, 28(31): 5055-5062. (in Chinese)
[24]	BOGDANOWICZ D R, LU H H. Multifunction co-culture model for evaluating cell-cell interactions. Methods in Molecular Biology. New York, NY: Springer New York, 2013: 29-36.
[25]	YU J N, SHI L H, WANG H, BILAN P J, YAO Z, SAMAAN M C, HE Q, KLIP A, NIU W Y. Conditioned medium from hypoxia-treated adipocytes renders muscle cells insulin resistant. European Journal of Cell Biology, 2011, 90(12): 1000-1015. doi: 10.1016/j.ejcb.2011.06.004 pmid: 21962636
[26]	CHOI S H, CHUNG K Y, JOHNSON B J, GO G W, KIM K H, CHOI C W, SMITH S B. Co-culture of bovine muscle satellite cells with preadipocytes increases PPARγ and C/EBPβ gene expression in differentiated myoblasts and increases GPR43 gene expression in adipocytes. The Journal of Nutritional Biochemistry, 2013, 24(3): 539-543.
[27]	LI A Q, SU X T, WANG Y N, CHENG G, ZAN L S, WANG H B. Effect of neudesin neurotrophic factor on differentiation of bovine preadipocytes and myoblasts in a co-culture system. Animals, 2020, 11(1): 34.
[28]	SU X T, WANG Y N, LI A Q, ZAN L S, WANG H B. Neudesin neurotrophic factor promotes bovine preadipocyte differentiation and inhibits myoblast myogenesis. Animals, 2019, 9(12): 1109.
[29]	张楚晗, 张东敏, 徐稳安. 牙髓再生组织工程中的细胞共培养体系. 中国组织工程研究, 2023, 27(15): 2379-2384.
	ZHANG C H, ZHANG D M, XU W A. Cell co-culture system of dental pulp regenerative tissue engineering. Chinese Journal of Tissue Engineering Research, 2023, 27(15): 2379-2384. (in Chinese)
[30]	NOURI-GOUSHKI M, EIJKEL B I M, MINNEBOO M, FRATILA- APACHITEI L E, ZADPOOR A A. Osteoimmunomodulatory potential of 3D printed submicron patterns assessed in a direct co-culture model. Biomaterials Advances, 2022, 139: 212993.
[31]	SAKTHIVEL K, O’BRIEN A, KIM K, HOORFAR M. Microfluidic analysis of heterotypic cellular interactions: a review of techniques and applications. TrAC Trends in Analytical Chemistry, 2019, 117: 166-185.
[32]	宋雅萍, 张久盘, 魏大为, 杨东梅, 赵毅昂, 姜超, 宋小雨, 吴昊. FOXO1基因调控牛前体脂肪细胞增殖和分化的功能研究. 中国农业大学学报, 2024, 29(4): 226-238.
	SONG Y P, ZHANG J P, WEI D W, YANG D M, ZHAO Y A, JIANG C, SONG X Y, WU H. Study on the function of FOXO1 gene in regulating the proliferation and differentiation of bovine preadipocytes. Journal of China Agricultural University, 2024, 29(4): 226-238. (in Chinese)
[33]	徐慧琳. 模拟肌内脂肪环境的方法构建及效果研究[D]. 武汉: 华中农业大学, 2022.
	XU H L. Method construction and effect study of simulating intramuscular fat environment[D]. Wuhan: Huazhong Agricultural University, 2022. (in Chinese)
[34]	KRUK Z A, BOTTEMA M J, REYES-VELIZ L, FORDER R E A, PITCHFORD W S, BOTTEMA C D K. Vitamin A and marbling attributes: Intramuscular fat hyperplasia effects in cattle. Meat Science, 2018, 137: 139-146. doi: S0309-1740(17)31152-X pmid: 29182958
[35]	KUPPUSAMY P, KIM D, SOUNDHARRAJAN I, HWANG I, CHOI K C. Adipose and muscle cell co-culture system: a novel in vitro tool to mimic the in vivo cellular environment. Biology, 2020, 10(1): 6.
[36]	MURPHY R A, MOORE S C, PLAYDON M, MEIRELLES O, NEWMAN A B, MILIJKOVIC I, KRITCHEVSKY S B, SCHWARTZ A, GOODPASTER B H, SAMPSON J, CAWTHON P, SIMONSICK E M, GERSZTEN R E, CLISH C B, HARRIS T B, STUDY F T H A. Metabolites associated with lean mass and adiposity in older black men. The Journals of Gerontology: Series A, 2017, 72(10): 1352-1359.
[37]	OZAKI K, SANO T, TSUJI N, MATSUURA T, NARAMA I. Carnitine is necessary to maintain the phenotype and function of brown adipose tissue. Laboratory Investigation, 2011, 91(5): 704-710. doi: 10.1038/labinvest.2011.6 pmid: 21321536
[38]	蒋金航. 牛PPAR γ基因调控脂肪细胞增殖和分化的机制研究[D]. 郑州: 河南农业大学, 2014.
	JIANG J H. The study of regulatory mechanisms of cattle ppar γ in adipocyte proliferation and differentiation[D]. Zhengzhou: Henan Agricultural University, 2014. (in Chinese)
[39]	宋雅萍, 雷召雄, 赵毅昂, 姜超, 王兴平, 罗仍卓么, 马云, 魏大为. 牛FoxO1基因CDS区克隆及其在脂肪细胞分化过程中的表达分析. 浙江农业学报, 2023, 35(5): 1016-1027. doi: 10.3969/j.issn.1004-1524.2023.05.05
	SONG Y P, LEI Z X, ZHAO Y A, JIANG C, WANG X P, LUORENGZHUOMA, MA Y, WEI D W. Cloning of CDS region of bovine FoxO1 gene and analysing expression pattern during adipocyte differentiation. Acta Agriculturae Zhejiangensis, 2023, 35(5): 1016-1027. (in Chinese)
[40]	SCHLOTTMANN I, EHRHART-BORNSTEIN M, WABITSCH M, BORNSTEIN S R, LAMOUNIER-ZEPTER V. Calcium-dependent release of adipocyte fatty acid binding protein from human adipocytes. International Journal of Obesity, 2014, 38(9): 1221-1227. doi: 10.1038/ijo.2013.241 pmid: 24352293
[41]	JANOT M, AUDFRAY A, LORIOL C, GERMOT A, MAFTAH A, DUPUY F. Glycogenome expression dynamics during mouse C2C12 myoblast differentiation suggests a sequential reorganization of membrane glycoconjugates. BMC Genomics, 2009, 10(1): 483.
[42]	秦瑞峰, 顾晓明. 生肌调节因子在肌肉发生和发育中的作用. 口腔颌面外科杂志, 2001, 11(3): 256-259, 274.
	QIN R F, GU X M. The role of myogenic regulatory factors in myogenesis and development. Chinese Journal of Oral & Maxillofacial Surgery, 2001, 11(3): 256-259, 274. (in Chinese)
[43]	王轶敏, 代阳, 刘新峰, 刘中伟, 李吉霞, 郭宏, 丁向彬. 牛骨骼肌卫星细胞的分离鉴定和诱导分化. 中国畜牧兽医, 2014, 41(7): 142-147.
	WANG Y M, DAI Y, LIU X F, LIU Z W, LI J X, GUO H, DING X B. Isolation, identification and induced differentiation of bovine skeletal muscle satellite cells. China Animal Husbandry & Veterinary Medicine, 2014, 41(7): 142-147. (in Chinese)
[44]	ASFOUR H A, ALLOUH M Z, SAID R S. Myogenic regulatory factors: The orchestrators of myogenesis after 30 years of discovery. Experimental Biology and Medicine, 2018, 243(2): 118-128.

基因 Gene	引物序列Primer sequence(5'→3')	产物大小 Product length (bp)	NCBI登录号 Genbank No.
PCNA	F: GAACCTCACCAGCATGTCCA	97	NM_001034494
PCNA	R: TACTAGTGCCAACGTGTCCG	97	NM_001034494
CDK2	F: GCCAGGAGTTACTTCTATGC	102	NM_001014934.1
CDK2	R: CTCCGTCCATCTTCATCC	102	NM_001014934.1
CCNE2	F：GCTTATGTCACTGATGGTGCTTG	122	NM_001015665.1
CCNE2	R：TTAGCCAGGAGATGACCGTTAC	122	NM_001015665.1
CCND1	F: TGGTCCTGGTGAACAAACTC	79	NM_176788
CCND1	R: ATCTGCTTGTTCTCCTCGGC	79	NM_176788
C/EBPβ	F: TTCCTCTCCGACCTCTTCTC	79	NM_176788
C/EBPβ	R: CCAGACTCACGTAGCCGTACT	79	NM_176788
FABP4	F: AAGTCAAGAGCATCGTAA	111	NM_174314.2
FABP4	R: CCAGCACCATCTTATCAT	111	NM_174314.2
PPARγ	F: AGGATGGGGTCCTCATATCC	121	NM_181024. 2
PPARγ	R: GCGTTGAACTTCACAGCAAA	121	NM_181024. 2
LPL	F: ACGATTATTGCTCAGCATGG	130	NM_001075120. 1
LPL	R: ACTTTGTACAGGCACAACCG	130	NM_001075120. 1
C/EBPα	F: TGGACAAGAACAGCAACGAG	130	NM_176784
C/EBPα	R: TTGTCACTGGTCAGCTCCAG	130	NM_176784
Myf6	F: ATTCCAGGGGGCTCGTGATA	107	NM_181811.2
Myf6	R:CGAGGAAATGCTGTCCACGA	107	NM_181811.2
Myf5	F: CCATCCGCTACATTGAGAGT	179	NM_174116.1
Myf5	R:GTAGACGCTGTCAAAACTGC	179	NM_174116.1
MyoG	F: CCAGTACATAGAGCGCCTGC	183	NM_001111325.1
MyoG	R: AGATGATCCCCTGGGTTGGG	183	NM_001111325.1
GAPDH	F:CCAACGTGTCTGTTGTGGAT	80	NM_001034034.2
GAPDH	R:CTGCTTCACCACCTTCTTGA	80	NM_001034034.2