油酰甘氨酸对C2C12成肌细胞增殖、分化和蛋白质沉积的影响  

The Effects of N-Oleoyl Glycine on the Proliferation, Differentiation and Protein Deposition of C2C12 Cells

作　　者：贺云朋 吴俊果 王松波[1] 王丽娜[1] 高萍[1] 束刚[1] 江青艳[1] 朱晓彤[1] 

机构地区：[1]华南农业大学动物科学学院,广州510642

出　　处：《农业生物技术学报》2016年第7期1054-1063,共10页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展计划(973)项目(No.2013CB127306和No.2012CB124701)

摘　　要：脂酰氨基酸是一类脂肪酸和氨基酸缩合生成的内源性化合物,在镇痛抗炎、细胞增殖和细胞凋亡等方面具有重要的调控作用。为探讨脂酰氨基酸对骨骼肌发育的影响,本研究以小鼠(Mus musculus)成肌细胞(myoblast cell line,C2C12)为对象,分别采用细胞计数检测法和肌酸激酶活性分析油酰甘氨酸(NOleoyl glycine,OLGly)对C2C12细胞的增殖和分化水平的影响。同时用q RT-PCR检测了增殖细胞核抗原、细胞周期素依赖性激酶抑制因子和生肌调节因子5、成肌素的m RNA表达水平。并且,本实验一方面测定了细胞总蛋白含量,另一方面采用Western blot法检测嘌呤霉素掺入水平,分析了蛋白质沉积相关蛋白以及蛋白质降解相关蛋白的表达水平。结果发现,与无水乙醇对照组相比,基础培养液中分别添加0.2、2和20μmol/L OLGly对C2C12细胞的数目、肌酸激酶活性以及增殖和分化标志基因表达水平均无显著影响;而蛋白质合成研究结果表明,OLGly可以剂量依赖性提高细胞总蛋白含量(P<0.05),此外,研究还发现,在OLGly处理组中,细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)蛋白和核糖体蛋白亚型6(ribosomal protein subunit 6,rp S6)蛋白的表达水平均呈现剂量和时间依赖性升高(P<0.05)。进一步研究发现,OLGly对蛋白质降解通路相关蛋白的表达无显著性影响。综合上述研究结果可见,OLGly能促进C2C12细胞蛋白质合成,但对其增殖和分化无明显影响。究其机制,可能与ERK和rp S6的蛋白表达水平的上调有关。研究结果为研制调控肌肉发育和蛋白质沉积的新型功能性调控物提供了实验依据。Fatty acyl amino acids, an endogenous compounds from the dehydration synthesis of fatty acids and amino acids, plays an important role of anti- inflammatory, cell proliferation and cell apoptosis. Skeletal muscle protein deposition is promoted by extracellular signal- regulated kinase（ERK） in mitogen- activated protein kinase（MAPK） signaling pathway and ribosomal protein（rp S6） in mammalian target of rapamycin（m TOR） signaling pathway, while muscle RING finger 1（Mu RF1） and muscle atrophy F- box（MAFBx） in forkheadbox O1（Fox O1） signaling pathway increase protein degradation. In addition, fatty acyl amino acids could be detected in skeletal muscle, which also express their receptors. To identify the effects of N- Oleoylglycine（OLGly） on the proliferation, differentiation and protein deposition of mouse（Mus musculus） myoblast cell line（C2C12）, cell counting kit-8（CCK-8） was used to calculate cell numbers and detected creatine kinase activity to identify the differentiation level of C2C12. Moreover, q RT- PCR was adopted to test the m RNA expression level of proliferating cell nuclear antigen（PCNA）, cell cycle protein dependent kinase inhibiting factors（p27 and p21）, myogenic detemination gene 5（Myf-5） and myogenic regulatory factor（My OG）, which were the marker genes of proliferation and differentiation of C2C12 cells. In this study, total protein content and the protein synthesis level（puromycin incorporation） were tested. Western blot was used to detect the expression level of m TOR, ERK, S6, Fox O1, MAFBx and Mu RF1. The results showed that 0.2, 2 or 20 μmol/L of OLGly had no significant effect on the cell numbers and the activity of creatine kinase, as well as the marker genes expression of proliferation and differentiation of C2C12 cells. Interestingly, OLGly could significantly enhance the protein contents of C2C12 myotubes in dose and time-dependent manners. 20 μmol/L of OLGly significantly enhanced the total contents of protein and the incorp

关 键 词：油酰甘氨酸 C2C12细胞 增殖分化 蛋白质沉积 信号通路 

分 类 号：S816.4[农业科学—饲料科学]