赖氨酸对苏尼特羊骨骼肌卫星细胞增殖、凋亡及分化的影响  

Effects of lysine on proliferation,apoptosis and differentiation ofskeletal muscle satellite cells in Sunit sheep

作　　者：王明旭 贺喜格 韩云飞 陈璐 吴金迪 格日勒图[1] WANG Mingxu;HE Xige;HAN Yunfei;CHEN Lu;WU Jindi;Gerelt(College of Food Science and Engineering,Inner Mongolia Agricultural University,Hohhot 010018,China;College of Life Science,Inner Mongolia University,Hohhot 010021,China)

机构地区：[1]内蒙古农业大学食品科学与工程学院,呼和浩特010018 [2]内蒙古大学生命科学学院,呼和浩特010021

出　　处：《黑龙江畜牧兽医》2025年第2期7-15,共9页Heilongjiang Animal Science And veterinary Medicine

基　　金：内蒙古自治区自然科学基金项目(2022QN03026);财政部和农业农村部:国家现代农业产业技术体系项目(CARS-38)。

摘　　要：为了探讨赖氨酸对肉羊骨骼肌卫星细胞(skeletal muscles satellite cells,SMSCs)增殖、凋亡及分化的影响,试验取胚胎期(3月龄)苏尼特羊背最长肌组织,对SMSCs进行分离、纯化、传代培养和鉴定,并用不同终浓度(0,1,2,4,8,16,32,64 mmol/L)的赖氨酸对SMSCs进行诱导,根据细胞存活率筛选最佳赖氨酸诱导浓度;取第4代SMSCs,设置两个组别(赖氨酸组和对照组),赖氨酸组在细胞中加入含最佳诱导浓度赖氨酸的完全培养基,对照组在细胞中加入不含有赖氨酸的完全培养基,于培养24,48,72,96,120 h后测定每孔OD_(450)值,考察赖氨酸对SMSCs增殖的影响;采用流式细胞术检测各组的细胞凋亡情况,考察赖氨酸对细胞凋亡的影响;采用实时荧光定量PCR方法测定成肌分化标志基因[成肌分化抗原(MYOD1)、肌细胞生成素(MYOG)、生肌因子5(MYF5)、生肌因子6(MYF6)、肌肉生长抑制素(MSTN)]和成脂分化标志基因[硬脂酰辅酶A去饱和酶(SCD)、脂肪酸结合蛋白(FABP4)]相对表达量,采用Western-blot方法检测MYOD1的蛋白表达水平,考察赖氨酸对细胞分化的影响。结果表明:经免疫荧光鉴定分离的细胞中配对盒7(Pax7)和MYOD1均呈阳性表达;最佳赖氨酸诱导浓度为4 mmol/L;培养24,48,72,96,120 h后,赖氨酸组的OD_(450)值均极显著高于对照组(P<0.01);培养24 h后,赖氨酸组细胞凋亡率和晚期凋亡率均极显著低于对照组(P<0.01),细胞中成肌分化标志基因(MYOD1、MYOG、MYF5和MYF6)和成脂分化标志基因(SCD、FABP4)相对表达量极显著或显著高于对照组(P<0.01或P<0.05),MSTN基因相对表达量显著低于对照组(P<0.05),MYOD1蛋白相对表达量极显著高于对照组(P<0.01)。说明赖氨酸可促进SMSCs增殖,抑制SMSCs凋亡,且可能通过调控SMSCs中成肌分化标志基因和蛋白及成脂分化标志基因的表达促进细胞分化。To investigate the effects of lysine on proliferation,apoptosis and differentiation of skeletal muscles satellite cells(SMSCs)of mutton sheep,the longissimus dorsi muscle tissues were collected from embryonic(3-month-old)Sunit sheep.SMSCs were isolated,purified,cultured and identified,and induced with different final concentrations of lysine(0,1,2,4,8,16,32,and 64 mmol/L).The optimal lysine induction concentration was selected according to cell viability.The fourth-generation SMSCs were divided into two groups:the lysine group,cultured with complete medium containing the optimal induced concentration of lysine,and the control group,cultured with complete medium without lysine.After 24,48,72,96 and 120 h of culture,the OD_(450) values of each well were measured to assess the effects of lysine on the SMSCs proliferation.Flow cytometry was used to evaluate the effect of lysine on apoptosis,real-time fluorescence quantitative PCR was used to detect the expression levels of myogenic differentiation marker genes(MYOD1,MYOG,MYF5,MYF6 and MSTN)and for lipid differentiation marker genes(SCD,FABP4),and Western-blot was used to detect the expression level of MYOD1,examining the effect of lysine on cell differentiation.The results showed that Pax7 and MYOD1 were positively expressed in the isolated cells identified by immunofluorescence.The optimal lysine induced concentration was 4 mmol/L.The OD_(450) values of lysine group were extremely significantly higher than those of the control group at all time points(P<0.01).After 24 h,the apoptosis rate and late apoptosis rate in lysine group were extremely significantly lower than those in the control group(P<0.01).The relative expression levels of myogenic differentiation marker genes(MYOD1,MYOG,MYF5 and MYF6)and lipid differentiation marker genes(SCD,FABP4)were extremely significantly higher or significantly higher than those·7·2025(02):7-15试验研究of control group(P<0.05),while the relative expression level of MSTN was significantly lower(P<0.01 or P<0.05).The relative e

关 键 词：苏尼特羊 骨骼肌卫星细胞 赖氨酸 细胞增殖 细胞凋亡 细胞分化 

分 类 号：S823.9[农业科学—畜牧学]