共培养体系下牛骨骼肌细胞对脂肪细胞的调控作用研究  

Research on the Regulatory Effects of Bovine Skeletal Muscle Cells on Adipocytes Under Co-Culture Conditions

作　　者：杨东梅[1] 张久盘 宋雅萍 宋小雨 姜超 马云 魏大为 YANG DongMei;ZHANG JiuPan;SONG YaPing;SONG XiaoYu;JIANG Chao;MA Yun;WEI DaWei(College of Animal Science and Technology Ningxia University/Key Laboratory of Ruminant Molecular Cell Breeding,Ningxia Hui Autonomous Region,Yinchuan 750021;Institute of Animal Science,Ningxia Academy of Agriculture and Forestry Sciences,Yinchuan 750021)

机构地区：[1]宁夏大学动物科技学院/宁夏回族自治区反刍动物分子细胞育种重点实验室,银川750021 [2]宁夏农林科学院动物科学研究所,银川750021

出　　处：《中国农业科学》2024年第18期3704-3718,共15页Scientia Agricultura Sinica

基　　金：“西部之光”人才培养计划(2024);国家自然科学基金(32060744,32202641);中央引导地方科技发展专项(2024FRD05052);动物科学国家一流本科专业;宁夏大学奶业现代产业学院资助项目。

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。【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 PPARγwas 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),whil

关 键 词：牛 脂肪细胞 骨骼肌细胞 共培养 细胞互作 

分 类 号：S823[农业科学—畜牧学]