长时间血清饥饿胁迫对猪骨骼肌卫星细胞代谢和自噬的影响  

Effects of Prolonged Serum Starvation Stress on Metabolism and Autophagy of Porcine Skeletal Muscle Satellite Cells

作　　者：高娟 范博钧 吴雅婕 王怡 杨跃飞[1,2] 鞠辉明 GAO Juan;FAN Bojun;WU Yajie;WANG Yi;YANG Yuefei;JU Huiming(College of Guangling,Yangzhou University,Yangzhou 225000,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis,College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学广陵学院,江苏扬州225000 [2]扬州大学兽医学院江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医杂志》2024年第2期32-38,共7页Chinese Journal of Veterinary Medicine

基　　金：江苏省自然科学基金项目(BK20201224);国家自然科学基金项目(31872323)。

摘　　要：为了探究长时间、不同程度饥饿胁迫条件对原代猪骨骼肌卫星细胞(SMSCs)代谢和自噬的影响,本试验通过控制SMSCs培养体系中血清浓度以形成不同程度饥饿胁迫,其中20%血清培养浓度为正常对照组,15%、10%、5%和0%血清培养浓度为试验组。细胞培养96 h后,通过流式细胞术检测各组细胞凋亡率、线粒体膜电位和活性氧(ROS)水平,通过ATP检测试剂盒测定细胞内ATP水平,通过Western blot检测各组细胞蛋白中自噬标志蛋白LC3b、p62以及AMPK-mTOR自噬信号通路中p-AMPK/AMPK和p-mTOR/mTOR蛋白的表达。结果显示,与20%血清组相比,0%血清和5%血清细胞凋亡率极显著上调(P<0.01),10%血清组细胞凋亡率显著上调(P<0.05);各试验组线粒体膜电位均极显著下降(P<0.01),ROS水平和ATP水平均极显著上调(P<0.01);细胞中LC3b蛋白的相对表达量随血清浓度降低而升高,p62蛋白的相对表达量随血清浓度降低而降低;p-mTOR/mTOR比值随血清浓度降低而降低,p-AMPK/AMPK比值随血清浓度降低而升高。结果表明,通过长期减少SMSCs培养体系中血清浓度,可以促进细胞代谢加快和凋亡发生,并且通过AMPK/mTOR信号通路激发细胞发生自噬,上述变化的程度与血清减少的程度呈正相关。本试验为后续探索不同程度饥饿胁迫对动物肌肉发育影响的研究提供理论依据。In order to explore the effects of prolonged and different degrees of serum starvation stress on metabolism and autophagy of primary porcine skeletal muscle satellite cells(SMSCs),this study controlled the serum concentration in the SMSCs culture system to create varying degrees of starvation stress.Specifically,20%serum concentration was used as the normal control group,while 15%,10%,5%,and 0%serum concentrations were used as experimental groups.After 96 hours of cell culture,the apoptosis rate,mitochondrial membrane potential,and reactive oxygen species(ROS)level of cells in each group were detected by flow cytometry.The intracellular ATP level was determined using an ATP assay kit.The expression of autophagy-related proteins LC3b,p62,and proteins in the AMPK-mTOR autophagy signaling pathway(p-AMPK/AMPK and p-mTOR/mTOR)was assessed by Western blot.The results showed that compared with the 20%serum group,the apoptosis rates of cells in the 0%and 5%serum groups were extremely significant upregulated(P<0.01),and the 10%serum group had a significantly increased apoptosis rate(P<0.05).The mitochondrial membrane potential of cells in all experimental groups extremely significant decreased(P<0.01),while ROS and ATP levels were extremely significant increased(P<0.01).The relative expression of LC3b protein in cells increased with decreasing serum concentration,while the relative expression of p62 protein decreased with decreasing serum concentration.The p-mTOR/mTOR ratio decreased with decreasing serum concentration,while the p-AMPK/AMPK ratio increased with decreasing serum concentration.The results suggest that by reducing the serum concentration in the SMSCs culture system for an extended period,cellular metabolism can be accelerated,apoptosis can occur,and autophagy can be induced through the AMPK/mTOR signaling pathway.The degree of these changes is positively correlated with the degree of serum reduction.This study provides a theoretical basis for further exploration of the impact of different degrees of starvati

关 键 词：血清饥饿 自噬 细胞代谢 AMPK/mTOR通路 骨骼肌卫星细胞 

分 类 号：S852.2[农业科学—基础兽医学]