FoxO1对牛骨骼肌细胞增殖、凋亡和分化的调控  

Study on the Role of FoxO1 in Regulating the Proliferation,Apoptosis and Differentiation of Bovine Skeletal Muscle Cells

作　　者：姜超 张久盘 宋雅萍 宋小雨 吴昊[1] 魏大为 JIANG Chao;ZHANG JiuPan;SONG YaPing;SONG XiaoYu;WU Hao;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6期1191-1203,共13页Scientia Agricultura Sinica

基　　金：宁夏自然科学基金(2023AAC03394);国家自然科学基金(32060744、32202641);宁夏重点研发计划项目(2023BCF01006);中国科学院“西部之光”人才培养计划项目(2023)。

摘　　要：【目的】骨骼肌是动物机体的重要组成成分,其生长发育直接影响畜禽肉产量,叉头转录因子O1(forkhead box protein O1,FoxO1)作为重要的转录调控因子,其与骨骼肌生长发育密切相关。探究过表达FoxO1对牛骨骼肌细胞增殖、凋亡与分化的作用,为肉牛遗传改良提供基础材料。【方法】采集牛的多个组织样品,提取其RNA并反转录,利用实时荧光定量PCR(qPCR)构建FoxO1组织表达谱。利用酶消化法分离得到牛骨骼肌细胞,通过观察其分化后肌管的形成以及qPCR检测其分化标志基因的表达情况来检验所分离细胞的分化性能。利用免疫荧光技术对牛骨骼肌细胞进行FoxO1亚细胞定位。设计并包装牛FoxO1过表达腺病毒,以提高牛骨骼肌细胞内FoxO1的表达。利用EdU染色检测过表达FoxO1对细胞相对增殖率的影响。利用流式细胞术检测过表达FoxO1对细胞周期分布的影响。利用qPCR检测过表达FoxO1对牛骨骼肌细胞增殖、凋亡和分化相关基因表达水平的影响。【结果】组织表达谱结果显示FoxO1在多个组织中均有表达,其在成年牛的背脂中表达量最高,在背最长肌组织中表达量最低,且FoxO1在犊牛背最长肌组织中的表达量要极显著高于成年牛的(P<0.01)。亚细胞定位结果显示FoxO1在牛骨骼肌细胞的细胞核和细胞质内均有表达,其细胞核内荧光强度高于细胞质。成功构建FoxO1过表达载体,并完成FoxO1重组过表达腺病毒的包装与扩繁,在感染牛骨骼肌细胞后,能显著提高FoxO1表达水平(P<0.01)。EdU检测显示过表达FoxO1会显著降低细胞增殖率(P<0.01),流式细胞周期检测显示过表达FoxO1会显著增加G1期细胞数并减少S期和G2期细胞数,抑制细胞G1/S期的转化,并减少G2期细胞的形成。利用qPCR进一步检测发现,增殖相关基因PCNA、CDK1、CDK2、CCNA2、CCNB1、CCND1和CCNE2均极显著下调(P<0.01),促凋亡相关基因BAD和BAX显著上调以及抑凋亡基�【Objective】Skeletal muscle is an important component of animal body,and its growth and development directly affect the meat yield of livestock and poultry.As an important transcription regulator,fork head box protein O1(FoxO1)is closely related to the growth and development of skeletal muscle.Exploring the effects of overexpression of FoxO1 gene on the proliferation,apoptosis and differentiation of bovine skeletal muscle cells could provide the theoretical basis for genetic improvement of beef.【Method】The tissue expression profile of FoxO1 was constructed by real-time fluorescence quantitative PCR(qPCR).To test the cell differentiation performance of bovine skeletal muscle cells which were isolated by enzyme digestion,the myotubes formation was observed and the expressions of differentiation marker genes were detected by qPCR.The subcellular localization of bovine skeletal muscle cells was carried out by immunofluorescence technique.In order to improve the expression of FoxO1 in bovine skeletal muscle cells,bovine FoxO1 overexpression adenovirus were designed and packaged.The effect of FoxO1 overexpression on the relative proliferation rate of cells was detected by EdU staining.Flow cytometry was used to detect the effect of FoxO1 overexpression on cell cycle distribution.The effects of FoxO1 overexpression on the expression levels of genes related to proliferation,apoptosis and differentiation of bovine skeletal muscle cells were investigated by qPCR.【Result】The results of tissue expression spectrum showed that FoxO1 was expressed in many tissues,with the highest expression in the back fat of adult cattle and the lowest expression in the longissimus dorsi muscle tissue,while the expression of FoxO1 in the longissimus dorsi muscle tissue of calves was significantly higher than that of adult cattle(P<0.01).The results of subcellular localization showed that FoxO1 was expressed in the nucleus and cytoplasm of bovine skeletal muscle cells,and the fluorescence intensity in the nucleus was higher than that

关 键 词：FOXO1 牛骨骼肌细胞 增殖 分化 

分 类 号：S823.2[农业科学—畜牧学]